Sample records for adenosine triphosphatase activity

  1. Localization of Adenosine Triphosphatase Activity on the Chloroplast Envelope in Tendrils of Pisum sativum1

    Sabnis, Dinkar D.; Gordon, Mildred; Galston, Arthur W.


    When samples of pea tendril tissue were incubated in the Wachstein-Meisel medium for the demonstration of adenosine triphosphatases, deposits of lead reaction product were localized between the membranes of the chloroplast envelope. The presence of Mg2+ was necessary for adenosine triphosphatase activity, and Ca2+ could not substitute for this requirement. Varying the pH of incubation to 5.5 or 9.4 inhibited enzyme activity, as did the addition of p-chloromercuribenzoic acid or N-ethylmaleimide. The adenosine triphosphatase was apparently inactivated or degraded when the plants were grown in the dark for 24 hours prior to incubation. The enzyme was substrate-specific for adenosine triphosphate; no reaction was obtained with adenosine diphosphate, uridine triphosphate, inosine triphosphate, p-nitrophenyl phosphate, and sodium β-glycerophosphate. Sites of nonspecific depositions of lead are described. The adenosine triphosphatase on the chloroplast envelope may be involved in the light-induced contraction of this organelle. Images PMID:4245003

  2. Effect in vitro of the Phenobarbital on the Activity of the Enzyme Adenosine Triphosphatase (ATPase of Sodium and Potassium Dependent in Human Placenta.

    María De Jesús Sánchez Bouza


    Full Text Available Background: Most of the drugs can pass to the fetus through the placenta, conditioning alterations of the development and fetal growth. Objective: To evaluate the effect in vitro of Phenobarbital on the activity of adenosine triphosphatase of sodium and potassium dependent in human placenta. Method: A study in vitro was carried out, in total homogenizes of placentas coming from 30 mothers that were assisted in the Gynecological and Obstetric Educational Provincial Hospital ¨Mariana Grajales¨, in Santa Clara, during the period March-July 1993. Phenobarbital was administered in concentrations of 0,1 mg/ml, 0,15 mg/ml and 0,2 mg/ml, keeping in mind the therapeutic dose to which was prescribed. The analyzed variables were: enzymatic activity and inhibition type. Results: The enzymatic activity of the adenosine triphosphatase of sodium and potassium dependent, diminished in a significant way, proportionally with the diminishment of the administered concentration of Phenobarbital. The inhibitory effect of the drug also turned out to be dependent of the concentration, presenting a major inhibition as the dose was increased. Conclusion: Phenobarbital produced highly significant decrease in the activity of adenosine triphosphatase of sodium and dependent potassium. This inhibition is of competitive type.

  3. Functional proteomics of adenosine triphosphatase system in the rat striatum during aging

    Roberto Federico Villa; Federica Ferrari; Antonella Gorini


    The maximum rates of adenosine triphosphatase (ATPase) systems related to energy consumption were systematically evaluated in synaptic plasma membranes isolated from the striata of male Wistar rats aged 2, 6, 12, 18, and 24 months, because of their key role in presynaptic nerve ending homeostasis. The following enzyme activities were evaluated: sodium-potassium-magnesium adenosine triphosphatase (Na+, K+, Mg2+-ATPase); ouabain-insensitive magnesium adenosine triphosphatase (Mg2+-ATPase); sodium-potassium adenosine triphosphatase (Na+, K+-ATPase); direct magnesium adenosine triphosphatase (Mg2+-ATPase); calcium-magnesium adenosine triphosphatase (Ca2+, Mg2+-ATPase); and acetylcholinesterase. The results showed that Na+, K+-ATPase decreased at 18 and 24 months, Ca2+, Mg2+-ATPase and acetylcholinesterase decreased from 6 months, while Mg2+-ATPase was unmodified. Therefore, ATPases vary independently during aging, suggesting that the ATPase enzyme systems are of neuropathological and pharmacological importance. This could be considered as an experimental model to study regeneration processes, because of the age-dependent modifications of specific synaptic plasma membranes. ATPases cause selective changes in some cerebral functions, especially bioenergetic systems. This could be of physiopathological significance, particularly in many central nervous system diseases, where, during regenerative processes, energy availability is essential.

  4. Oxidative stress parameters and erythrocyte membrane adenosine triphosphatase activities in streptozotocin-induced diabetic rats administered aqueous preparation of Kalanchoe Pinnata leaves

    Nikhil Menon


    Full Text Available Background: Diabetes mellitus is a chronic metabolic disease that according to the World Health Organization affects more than 382 million people. The rise in diabetes mellitus coupled with the lack of an effective treatment has led many to investigate medicinal plants to identify a viable alternative. Objective: To evaluate red blood cell (RBC membrane adenosine triphosphatase (ATPase activities and antioxidant levels in streptozotocin-induced diabetic rats administered aqueous preparation of Kalanchoe pinnata leaves. Materials and Methods: Diabetes mellitus was induced in rats by a single administration of streptozotocin (60 mg/kg. Diabetic rats were then treated with aqueous K. pinnata preparation (three mature leaves ~ 9.96 g/70 kg body weight or about 0.14 g/kg body weight/day for 30 days. Serum glucose, RBC membrane ATPase activities, and antioxidant levels were determined. Results: We noted weight loss and reduced food consumption in the treated diabetic group. Serum glucose levels were reduced in the treated diabetic group compared to the other groups. Superoxide dismutase activity and glutathione levels were not significantly elevated in the treated group compared to the diabetic group. However, serum catalase activity was significantly (P < 0.05 increased in the treated diabetic group compared to the other groups. Serum thiobarbituric acid reactive substances were not significantly altered among the groups. There was a significant (P < 0.05 increase in Mg2+ ATPase activity and a nonsignificant increase in Na+/K+ ATPase activity in the RBC membrane of the treated diabetic group compared to the diabetic group. Conclusion: The consumption of aqueous preparation of K. pinnata may accrue benefits in the management of diabetes by lowering oxidative stress often associated with the disease and improving the availability of cellular magnesium through an increase in the magnesium ATPase pump in the RBC membrane for increased cellular metabolism

  5. [Thiamine triphosphatase activity in mammalian mitochondria].

    Rusina, I M; Makarchikov, A F


    Mitochondrial preparations isolated from bovine kidney and brain as well as the liver and the brain of rat show thiamine triphosphatase (ThTPase) activity. The activity was determined from the particles by freezing-thawing suggesting that a soluble enzyme is involved. The liberation patterns of ThTPase and marker enzyme activities from mitochondria under osmotic shock or treatment with increasing Triton X-100 concentrations indicate the presence of ThTPase both in the matrix and intermembrane space. It was found, basing on gel filtration behavior, that the mitochondrial ThTPase has the same molecular mass as specific cytosolic ThTPase (EC The enzymes, however, were clearly distinguishable in Km values, the mitochondrial one showing a higher apparent affinity for substrate. These results imply the existence of ThTPase multiple forms in mammalian cells.

  6. Glucocorticoid Regulation of Rat Renal Sodium Potassium Adenosine Triphosphatase


    response is not mediated by de novo protein synthesis but is secondary to enhanced sodium influx. The osmotic diuresis seen in uncontrolled...mediated increase in renal NaK-ATPase activity. The administration of anti-insulin serum caused a brisk natriuresis in control rat kidneys while having...hydrocortisone on water diuresis and renal function in man. J. Clin. Invest. 36: 767-779, 1957. Rane, S. and A. Aperia. Ontogeny of Na-K-ATPase

  7. Reduction of nitrates in Cucumis sativus L. seedlings II. Influence of tungsten and vanadium on nitrate reductase and adenosine triphosphatase activities

    Józef Buczek


    Full Text Available ATPases isolated from the roots of cucumber seedlings activated by Mg+2 ions in experiments in vitro, were fairly distinctly inhibited by Ca-2 ions, very slightly inhibited by fluorides and molybdenum ions while NO3- anions had no effect on the level of ATPase activity studied. Introduction into the nutrient of 10-4 M Na2WO4 or 10-3 M Na VO3 (inhibitors of nitrate reductase NR distinctly inhibited activity of the ATPase under study especially of fractions IIa and III, and inhibited NR activity and lowered uptake of NO3-. WO4-2 and VO3 inhibited to the same extent absorption and reduction of NO3- in the initial phase of NR induction, whereas at a later stage both inhibitors checked reduction to a greater degree than uptake of NO3-. The results indicate the possibility of certain ATPase participation in assimilating nitrates, and suggest that in the initial stage of biosynthesis of the NR enzyme system, activity of the enzyme is distinctly dependent upon NO3- transport and the level of NR activity limited by the amount of nitrate taken up. At a later an additional mechanism of NO3- transport probably functions, not connected with simultaneous reduction of nitrates. On the basis of results the Butz and Jackson (1977 hypothesis concerning a model for the absorption and reduction of NO3- by plant tissues is discussed.

  8. Tegumental Ca-stimulated adenosine triphosphatase activity in adult Schistosoma mansoni worms Atividade da adenosina trifosfatase estimulada pelo Ca no tegumento de vermes adultos de Schistosoma mansoni

    Italo M. Cesari


    Full Text Available A Ca-stimulated ATPase activity (pH 9.5 associated with the tegumental membrane enriched (TME fraction of Schistosoma mansoni adults was partially inhibited by NAP-taurine or by increasing concentrations of chlorpromazine; endogenous calmodulin was found associated with the TME fraction. A similar activity (pH 8.6 was histochemically visualized whithin the tegument of fixed worms on the cytoplasmic leaflet of both the doubel surface membrane and the basement membrane; this reaction was inhibited by 1 µM chloropromazine and it was also observed on the inner side of double membrane vesicles present in the TME fraction. No ATPase activity could be seen at alkaline pH with added Mg or Na/K ions. Without ATP, the addition of external Ca to the fixed worms induced the appearance of lead precipitates on the tegumental discoid bodies; this reaction was inhibited by molybdate and not by chlorpromazine. The intrategumentary regulation of calcium by the systems described and the possible use of phenothiazines against schistosimes are discussed.A atividade ATPse (pH 9.5 estimulada por ions de Ca associados a uma fração enriquecida de membranas do tegumento (fração EMT de vermes adultos de Schistosoma mansoni, foi inibida pro NAP-taurina ou por concentrações crescentes de clorpromacina. Foi encontrada calmodulina enfogena associada principlamente a esta fração. Em vermes adultos fixados com glutaraldeido se detectou histoquimicamente uma atividade ATPase similar (pH 8.6 na face citoplasmática da dupla membrana de superfície e da membrana por 1 µM de clorpromacina e foi também observada na face interna de vesículas de dupla membrana presentes na fração EMT. Não se pôde detectar atividade ATpase em pH alcalino na presença de ions de Mg ou Na/K. A adição externa de Ca, sem ATP, aos vermes fixados induz ao aparecimento de precipitados nos corpos discóides do tegumento; esta reação foi inibida. Os resultados são discutidos em relação a

  9. Effect in vitro of the Phenobarbital on the Activity of the Enzyme Adenosine Triphosphatase (ATPase of Sodium and Potassium Dependent in Human Placenta. Efecto in vitro del fenobarbital sobre la actividad de la enzima Adenosintrifosfatasa de sodio y potasio dependiente en placenta humana.

    Pedro Sánchez Frenes


    Full Text Available

    Background: Most of the drugs can pass to the fetus through the placenta, conditioning alterations of the development and fetal growth. Objective: To evaluate the effect in vitro of Phenobarbital on the activity of adenosine triphosphatase of sodium and potassium dependent in human placenta. Method: A study in vitro was carried out, in total homogenizes of placentas coming from 30 mothers that were assisted in the Gynecological and Obstetric Educational Provincial Hospital ¨Mariana Grajales¨, in Santa Clara, during the period March-July 1993. Phenobarbital was administered in concentrations of 0,1 mg/ml, 0,15 mg/ml and 0,2 mg/ml, keeping in mind the therapeutic dose to which was prescribed. The analyzed variables were: enzymatic activity and inhibition type. Results: The enzymatic activity of the adenosine triphosphatase of sodium and potassium dependent, diminished in a significant way, proportionally with the diminishment of the administered concentration of Phenobarbital. The inhibitory effect of the drug also turned out to be dependent of the concentration, presenting a major inhibition as the dose was increased. Conclusion: Phenobarbital produced highly significant decrease in the activity of adenosine triphosphatase of sodium and dependent potassium. This inhibition is of competitive type.

    Fundamento: La mayoría de los fármacos, en mayor o menor medida, son capaces de pasar al feto a través de la placenta, condicionando alteraciones del desarrollo y del crecimiento fetal. Objetivo: Evaluar el efecto in vitro del fenobarbital sobre la actividad de la Adenosintrifosfatasa de sodio y potasio dependiente de placenta humana. Método: Se realizó un estudio in vitro, en homogeneizados totales de placentas provenientes de 30 madres que fueron atendidas en el Hospital Provincial Docente Ginecobst

  10. Insecticidal properties of essential oils against Tribolium castaneum (Herbst) and their inhibitory effects on acetylcholinesterase and adenosine triphosphatases.

    Abou-Taleb, Hamdy K; Mohamed, Magdy I E; Shawir, Mohamed S; Abdelgaleil, Samir A M


    Essential oils from 20 Egyptian plants were obtained by using hydrodistillation. The chemical composition of the isolated oils was identified by gas chromatograph/mass spectrometer. Fumigant and contact toxicities of the essential oils were evaluated against the adults of Tribolium castaneum. In fumigation assays, the oil of Origanum vulgare (LC50 = 9.97 mg/L air) displayed the highest toxicity towards the adults of T. castaneum. In contact assays, the oils of Artemisia monosperma (LC50 = 0.07 mg/cm(2)) and O. vulgare (LC50 = 0.07 mg/cm(2)) were the most potent toxicants against the adults of T. castaneum. Biochemical studies showed that the tested oils caused pronounced inhibition of acetylcholinesterase (AChE) and adenosine triphosphatases (ATPases) isolated from the larvae of T. castaneum. The oil Cupressus macrocarpa (IC50 = 12.3 mg/L) was the most potent inhibitor of AChE, while the oil of Calistemon viminals (IC50 = 4.4 mg/L) was the most potent inhibitor of ATPases.

  11. Alteration of erythrocyte deformability and adenosine triphosphatase activity of cell membrana in essential hypertensive patients complicated with cerebral infarction%高血压病并发脑梗死患者红细胞变形性及细胞膜ATP酶活性的研究

    史瑞英; 周宏研; 孙明


    目的:研究高血压病(EH)及并发脑梗死(HCI)患者红细胞变形性(RCD)及细胞膜ATP酶活性的改变。方法:采用粘度法、化学比色法测定了20例正常人、30例EH及30例HCI患者的红细胞变形指数(DI)、红细胞膜Na+,K+-ATP酶和Ca2+-ATP酶的活性。结果:(1) EH组与正常对照组相比,DI值显著升高,ATP酶活性降低,EH患者DI与ATP酶活性呈负相关,与DBP呈正相关;(2)HCI组与EH组相比,DI显著升高,ATP酶活性显著降低;HCI患者DI与ATP酶活性呈负相关。结论:(1) EH患者RCD降低,并与细胞膜ATP酶活性降低及血压升高相关;(2)RCD降低可能与EH患者脑梗死的发生有关。%Objective: To investigate the alteration of red cell deformability (RCD) and adenosine triphosphatase (ATPase) activity of cell membrane in essential hypertensive (EH) patients and essential hypertension patients complicated with cerebral infarction (HCI). Methods: Twenty healthy individuals, 30 EH patients and 30 HCI patients were selected as subjects. The erythrocyte deformability indexes (DI)、 Na+, K+-ATPase and Ca2+ATPase activities were examined. Results: (1) EH patients had significantly higher DI, lower ATPase activity as compared to healthy individuals. There were significantly negative correlation between DI and ATPase activity, and a significantly positive correalation between DI and diastolic blood pressure in EH patients; (2) There was significantly higher DI, lower ATPase activity in HCI patients than those in EH patients. There was significantly negative correlation between DIand ATPase activity in HCI patients. Conclusion: (1) RCD decreases in EH patients, and RCD is correlated to the decreased ATPase of cell membrane and elevated blood pressure; (2) The decreased RCD level may be related to the occurrence of HCI.

  12. D-Pinitol attenuates 7, 12 dimethylbenz [a] anthracene induced hazards through modulating protein bound carbohydrates, adenosine triphosphatases and lysosomal enzymes during experimental mammary carcinogenesis.

    Rengarajan, Thamaraiselvan; Nandakumar, Natarajan; Balasubramanian, Maruthaiveeran Periyasamy


    We have reported here that the ameliorative potentials of D-Pinitol during 7, 12-Dimethylbenz [a] anthracene induced experimental breast carcinogenesis. DMBA is a potent organ specific carcinogen which is widely employed to induce mammary carcinoma in rats. D-Pinitol a natural inositol has been reported to found in soybean with many biological functions. The female sprague dawley rats were subjected to carcinogen 7, 12-DMBA and the ameliorative potentials of dietary compound D-Pinitol was investigated with reference to cell surface glycoproteins, lysosomal enzymes and adenosine triphosphatases. Interestingly, administration of D-Pinitol was found to be significantly down regulated the breast tissue glycoproteins and lysosomal enzymes and in contrast the levels of adenosine triphosphatases were remarkably up regulated. Further, the biochemical changes were well reflected and evidenced in the histology of breast and liver tissues. Thus, it can be concluded from the present study that D-Pinitol efficiently attenuates the hazardous consequences of the environmental carcinogen 7,12-DMBA through modulating cell surface glycoproteins, membrane protective role both in lysosomal and ATPase compartment via its antioxidant nature which ultimately results in the findings of future innovative remedies for genotoxin mediated hazards.

  13. Method for isolation of Escherichia coli mutants with defects in the proton-translocating sector of the membrane adenosine triphosphatase complex.

    Fillingame, R H; Knoebel, K; Wopat, A E


    A technique for selecting mutants of Escherichia coli in which the proton-translocating sector of the adenosine triphosphatase (ATPase) complex has been inactivated is reported. The procedure uses a strain of E. coli (NR-70) lacking the extrinsic (F1) sector of the ATPase complex and which in consequently permeable to protons (B. P. Rosen, J. Bacteriol. 116:1124--1129, 1973). After growing strain NR-70 under noninducing conditions for the lac operon, cells were mutagenized and plated on minimal medium containing low concentrations of lactose. Several mutants of strain NR-70 were isolated as large colonies on these plates, apparently because they could concentrate lactose more efficiently. A description of one of the mutants, strain KW-1, is reported here. The most distinguishing difference in growth properties of the two strains was that, when transferred to medium containing low concentrations of lactose, strain KW-1 induced the lac operon with a shorter lag time than strain NR-70. The mutation in strain KW-1 leading to more rapid growth on lactose was cotransducible with the asn and unc loci, at 83 min on the E. coli genetic map. Intact cells of strain KW-1 actively transported L-proline as well as did wild-type cells, whereas cells of strain NR-70 were markedly deficient in L-proline transport. The improvement in the transport capacity of strain KW-1 correlated with a marked decrease in proton permeability relative to that of strain NR-70. Based on an acid-base pulse technique that measured the proton conductance of the membranes of intact cells, strain NR-70 was at least 10 times more permeable to protons than was the wild type, whereas strain KW-1 was only 2 times more permeable. The transport properties and proton conductance were also compared with membrane vesicles prepared by osmotic shock. With either D-lactate or ascorbate-N-methylphenazonium methosulfate as respiratory substrates, vesicles of strain KW-1 transported L-proline much more rapidly than did

  14. Guanosine triphosphatase activating protein (GAP) interacts with the p21 ras effector binding domain

    Adari, H; Lowy, D R; Willumsen, B M;


    A cytoplasmic protein that greatly enhances the guanosine triphosphatase (GTPase) activity of N-ras protein but does not affect the activity of oncogenic ras mutants has been recently described. This protein (GAP) is shown here to be ubiquitous in higher eukaryotes and to interact with H-ras as w......A cytoplasmic protein that greatly enhances the guanosine triphosphatase (GTPase) activity of N-ras protein but does not affect the activity of oncogenic ras mutants has been recently described. This protein (GAP) is shown here to be ubiquitous in higher eukaryotes and to interact with H......-ras as well as with N-ras proteins. To identify the region of ras p21 with which GAP interacts, 21 H-ras mutant proteins were purified and tested for their ability to undergo stimulation of GTPase activity by GAP. Mutations in nonessential regions of H-ras p21 as well as mutations in its carboxyl....... Transforming mutations at positions 12, 59, and 61 (the phosphoryl binding region) abolished GTPase stimulation by GAP. Point mutations in the putative effector region of ras p21 (amino acids 35, 36, and 38) were also insensitive to GAP. However, a point mutation at position 39, shown previously not to impair...

  15. Assembly of the adenosine triphosphatase complex in Escherichia coli: assembly of F0 is dependent on the formation of specific F1 subunits.

    Cox, G B; Downie, J A; Langman, L; Senior, A E; Ash, G; Fayle, D R; Gibson, F


    A strain of Escherichia coli (AN1007) carrying the polar uncD436 allele which affects the operon coding for the F1-F0 adenosine triphosphatase (ATPase) complex was isolated and characterized. The uncD436 allele affected the two genes most distal to the operon promoter, i.e., uncD and uncC. Although the genes coding for the F0 portion of the ATPase complex were not affected in strains carrying this mutant allele, the lack of reconstitution of washed membranes by normal F1 ATPase suggested that a functional F0 might not be formed. This conclusion was supported by the observation that the 18,000-molecular-weight F0 subunit, coded for by the uncF gene, was absent from the membranes. Plasmid pAN36 (uncD+C+), when inserted into a strain carrying the uncD436 allele, resulted in the incorporation of the 18,000-molecular-weight F0 subunit into the membrane. A further series of experiments with Mu-induced polarity mutants, with and without plasmid pAN36, showed that the formation of both the alpha- and beta-subunits of F1 ATPase was an essential prerequisite to the incorporation into the membrane of the 18,000-molecular-weight F0 subunit and to the formation of a functional F0. Examination of the polypeptide composition of membranes from various unc mutants allowed a sequence for the normal assembly of the F1-F0 ATPase complex to be proposed.

  16. High inorganic triphosphatase activities in bacteria and mammalian cells: identification of the enzymes involved.

    Gregory Kohn

    Full Text Available BACKGROUND: We recently characterized a specific inorganic triphosphatase (PPPase from Nitrosomonas europaea. This enzyme belongs to the CYTH superfamily of proteins. Many bacterial members of this family are annotated as predicted adenylate cyclases, because one of the founding members is CyaB adenylate cyclase from A. hydrophila. The aim of the present study is to determine whether other members of the CYTH protein family also have a PPPase activity, if there are PPPase activities in animal tissues and what enzymes are responsible for these activities. METHODOLOGY/PRINCIPAL FINDINGS: Recombinant enzymes were expressed and purified as GST- or His-tagged fusion proteins and the enzyme activities were determined by measuring the release of inorganic phosphate. We show that the hitherto uncharacterized E. coli CYTH protein ygiF is a specific PPPase, but it contributes only marginally to the total PPPase activity in this organism, where the main enzyme responsible for hydrolysis of inorganic triphosphate (PPP(i is inorganic pyrophosphatase. We further show that CyaB hydrolyzes PPP(i but this activity is low compared to its adenylate cyclase activity. Finally we demonstrate a high PPPase activity in mammalian and quail tissue, particularly in the brain. We show that this activity is mainly due to Prune, an exopolyphosphatase overexpressed in metastatic tumors where it promotes cell motility. CONCLUSIONS AND GENERAL SIGNIFICANCE: We show for the first time that PPPase activities are widespread in bacteria and animals. We identified the enzymes responsible for these activities but we were unable to detect significant amounts of PPP(i in E. coli or brain extracts using ion chromatography and capillary electrophoresis. The role of these enzymes may be to hydrolyze PPP(i, which could be cytotoxic because of its high affinity for Ca(2+, thereby interfering with Ca(2+ signaling.

  17. New member of the guanosine triphosphatase activating protein family in the human epididymis

    Xiangqi Li; Qiang Liu; Shigui Liu; Jinsong Zhang; Yonglian Zhang


    The effect of the guanosine triphosphatase activating proteins (GAPs) on spermatogenesis has been studied for years,though no GAPs have been explored in epididymis, an essential organ for sperm maturation. In this study, a new GAP member, designated as MacGAP, was cloned in human epididymis. The MacGAP gene encodes a protein of 618 amino acids with a putative size of 70 kDa and harbors the conservedRhoGAPdomain. The N-terminal and C-terminal peptides of MacGAP were expressed and their corresponding antiserawere prepared. The antisera against N-terminal peptide could detect antigen as low as 0.3 ng, and its specificity was alsoconfirmed. However, the antisera against C-terminal peptide failed to detect its antigen because of its low sensitivity.Immunohistochemistry showed that the MacGAP protein was dependent on epididymis and had a region-specific expression pattern, with high expression in the epithelial cells' basal section in the caput region. The results have created a foundation for further interpretation of the biological effects of GAPs in sperm maturation.

  18. NTPase and 5'-RNA triphosphatase activities of Chikungunya virus nsP2 protein.

    Karpe, Yogesh A; Aher, Pankaj P; Lole, Kavita S


    Chikungunya virus (CHIKV) is an insect borne virus (genus: Alphavirus) which causes acute febrile illness in humans followed by a prolonged arthralgic disease that affects the joints of the extremities. Re-emergence of the virus in the form of outbreaks in last 6-7 years has posed a serious public health problem. CHIKV has a positive sense single stranded RNA genome of about 12,000 nt. Open reading frame 1 of the viral genome encodes a polyprotein precursor, nsP1234, which is processed further into different non structural proteins (nsP1, nsP2, nsP3 and nsP4). Sequence based analyses have shown helicase domain at the N-terminus and protease domain at C-terminus of nsP2. A detailed biochemical analysis of NTPase/RNA helicase and 5'-RNA phosphatase activities of recombinant CHIKV-nsP2T protein (containing conserved NTPase/helicase motifs in the N-terminus and partial papain like protease domain at the C-terminus) was carried out. The protein could hydrolyze all NTPs except dTTP and showed better efficiency for ATP, dATP, GTP and dGTP hydrolysis. ATP was the most preferred substrate by the enzyme. CHIKV-nsP2T also showed 5'-triphosphatase (RTPase) activity that specifically removes the γ-phosphate from the 5' end of RNA. Both NTPase and RTPase activities of the protein were completely dependent on Mg(2+) ions. RTPase activity was inhibited by ATP showing sharing of the binding motif by NTP and RNA. Both enzymatic activities were drastically reduced by mutations in the NTP binding motif (GKT) and co-factor, Mg(2+) ion binding motif (DEXX) suggesting that they have a common catalytic site.

  19. NTPase and 5'-RNA triphosphatase activities of Chikungunya virus nsP2 protein.

    Yogesh A Karpe

    Full Text Available Chikungunya virus (CHIKV is an insect borne virus (genus: Alphavirus which causes acute febrile illness in humans followed by a prolonged arthralgic disease that affects the joints of the extremities. Re-emergence of the virus in the form of outbreaks in last 6-7 years has posed a serious public health problem. CHIKV has a positive sense single stranded RNA genome of about 12,000 nt. Open reading frame 1 of the viral genome encodes a polyprotein precursor, nsP1234, which is processed further into different non structural proteins (nsP1, nsP2, nsP3 and nsP4. Sequence based analyses have shown helicase domain at the N-terminus and protease domain at C-terminus of nsP2. A detailed biochemical analysis of NTPase/RNA helicase and 5'-RNA phosphatase activities of recombinant CHIKV-nsP2T protein (containing conserved NTPase/helicase motifs in the N-terminus and partial papain like protease domain at the C-terminus was carried out. The protein could hydrolyze all NTPs except dTTP and showed better efficiency for ATP, dATP, GTP and dGTP hydrolysis. ATP was the most preferred substrate by the enzyme. CHIKV-nsP2T also showed 5'-triphosphatase (RTPase activity that specifically removes the γ-phosphate from the 5' end of RNA. Both NTPase and RTPase activities of the protein were completely dependent on Mg(2+ ions. RTPase activity was inhibited by ATP showing sharing of the binding motif by NTP and RNA. Both enzymatic activities were drastically reduced by mutations in the NTP binding motif (GKT and co-factor, Mg(2+ ion binding motif (DEXX suggesting that they have a common catalytic site.

  20. Neurofibromatosis: The role of guanosine triphosphatase activating proteins in sensory neuron function

    Cynthia M. Hingtgen


    Neurofibromatosis type 1 (NF1) is a common autosomal dominant disease characterized by formation of multiple benign and malignant tumors. People with this disorder also experience chronic pain, which can be disabling. Neurofibromin, the protein product of the Nfl gene, is a gnanosine triphosphatase activating protein (GAP) for p21Ras (Ras). Loss of Nfl results in an increase in activity of the Ras transduction cascade. Because of the growing evidence suggesting involvement of downstream components of the Ras transduction cascade in the sensitization of nociceptive sensory neurons, we examined the stimulus-evoked release of the neuropeptides, substance P (SP) and calcitonin gene-related peptide (CGRP), from primary sensory neurons of mice with a mutation of the Nfl gene (NfI+1-). Measuring the levels of SP and CGRP by radioimmunoassay, we demonstrated that capsaicin-stimulated release of neuropep-tides is 3-5 folds higher in spinal cord slices from Nfl+1-mice than that from wildtype mouse tissue. In addition, the potassium- and capsaicin-stimulated release of CGRP from the culture of sensory neurons isolated from Nfl+1- mice was more than double that from the culture of wildtype neurons. Using patch-clamp electrophysiological techniques, we also examined the excitability of capsaicin-sensitive sensory neurons. It was found that the number of action potentials generated by the neurons from Nfl+1- mice, responsing to a ramp of depolarizing current, was more than three times of that generated by wildtype neurons. Consistent with that observation, neurons from Nfl+1- mice had lower firing thresholds, lower rheobase currents and shorter firing latencies compared with wildtype neurons. These data clearly demonstrate that GAPs, such as neurofihromin, can alter the excitability of nociceptive sensory neurons. The augmented response of sensory neurons with altered Ras signaling may explain the abnormal pain sensations experienced by people with NFI and suggests an important

  1. Homeostatic control of synaptic activity by endogenous adenosine is mediated by adenosine kinase.

    Diógenes, Maria José; Neves-Tomé, Raquel; Fucile, Sergio; Martinello, Katiuscia; Scianni, Maria; Theofilas, Panos; Lopatár, Jan; Ribeiro, Joaquim A; Maggi, Laura; Frenguelli, Bruno G; Limatola, Cristina; Boison, Detlev; Sebastião, Ana M


    Extracellular adenosine, a key regulator of neuronal excitability, is metabolized by astrocyte-based enzyme adenosine kinase (ADK). We hypothesized that ADK might be an upstream regulator of adenosine-based homeostatic brain functions by simultaneously affecting several downstream pathways. We therefore studied the relationship between ADK expression, levels of extracellular adenosine, synaptic transmission, intrinsic excitability, and brain-derived neurotrophic factor (BDNF)-dependent synaptic actions in transgenic mice underexpressing or overexpressing ADK. We demonstrate that ADK: 1) Critically influences the basal tone of adenosine, evaluated by microelectrode adenosine biosensors, and its release following stimulation; 2) determines the degree of tonic adenosine-dependent synaptic inhibition, which correlates with differential plasticity at hippocampal synapses with low release probability; 3) modulates the age-dependent effects of BDNF on hippocampal synaptic transmission, an action dependent upon co-activation of adenosine A2A receptors; and 4) influences GABAA receptor-mediated currents in CA3 pyramidal neurons. We conclude that ADK provides important upstream regulation of adenosine-based homeostatic function of the brain and that this mechanism is necessary and permissive to synaptic actions of adenosine acting on multiple pathways. These mechanistic studies support previous therapeutic studies and implicate ADK as a promising therapeutic target for upstream control of multiple neuronal signaling pathways crucial for a variety of neurological disorders.

  2. Inhibition of transducin activation and guanosine triphosphatase activity by aluminum ion.

    Miller, J L; Hubbard, C M; Litman, B J; Macdonald, T L


    Aluminum ion perturbs the activity of a number of physiologically important enzymes, including members of a family of guanine nucleotide-binding proteins (G-proteins). G-proteins couple cellular receptor proteins to a variety of effector enzymes (including adenylate cyclase, phospholipase C, and the rod photoreceptor phosphodiesterase). We show herein that subnanomolar concentrations of free aluminum ion, produced in a carefully defined and kinetically stable manner through the buffering of total aluminum at 0.1-1.0 mM with calculated ratios of chelating agents, inhibit both the receptor-mediated activation and the self-inactivating GTPase activity of the rod photoreceptor G-protein, Gv. In the presence of 4 X 10(-10) M free aluminum ion, GTPase activity is inhibited from about 25-60% as the magnesium ion concentration is reduced from 10(-3) to about 5 X 10(-5) M. The principal effect of aluminum ion upon Gv is to inhibit receptor catalyzed nucleotide exchange. Binding of the GTP analog 5'-guanylyl imidodiphosphate can be reduced by as much as 90% by aluminum ion following subsaturating rhodopsin stimulation. Aluminum ion can produce either competitive or mixed noncompetitive inhibition of rhodopsin-catalyzed Gv activation and GTPase activity, as a function of whether Gv undergoes single (competitive), or multiple (mixed noncompetitive) nucleotide exchanges. The rod photoreceptor phosphodiesterase is only slightly inhibited by similar aluminum ion activities. Light- and Gv-coupled phosphodiesterase activation exhibits both a lower maximum rate of cyclic guanosine monophosphate hydrolysis and a slower inactivation in the presence of aluminum ion activities from about 10(-12) - 10(-10) M. These data suggest that intracellular free aluminum ion concentrations in the subnanomolar range could markedly affect the ability of cells to transduce extracellular signals. Interestingly, the combination of Al3+ and F- to produce the fluoro-aluminate species (AlFx) also inhibits

  3. Properties of mammalian nuclear-envelope nucleoside triphosphatase.

    Agutter, P S; Cockrill, J B; Lavine, J E; McCaldin, B; Sim, R B


    The nucleoside triphosphatase activities of the nuclear envelopes from rat liver, pig liver and simian-virus-40-transformed mouse-embryo 3T3 cells were shown to exhibit similar parperties. All three preparations hydrolyse ATP, 2'-dATP, 3'-dATP, GTP, CTP and UTP in the presence of Mg2+, Ca2+, Mn2+ and Co2+ with a pH optimum of 8.0, are sensitive to inhibition by mercurials, arsenicals, quercetin, proflavin and adenosine 5'-[gamma-thio]triphosphate and are partially inactivated by exposure to high ionic strength. The kinetic behaviour is similar for all substrates irrespective of the source of material. The typical Eadie-Hofstee plot, which is concave upwards at pH 8.0 when the ionic strength is 20mM, becomes linear when the pH is increased to 8.5 or the ionic strength to 160mM. The overall evidence, particularly the labelling of only one polypeptide by [gamma-32P]ATP, suggests that under the conditions of preparation and assay used only one class of nucleoside triphosphatase active sites is detectable in nuclear envelopes. The importance of these results for an understanding of the role of the enzyme in vivo is discussed.

  4. N6-(2-Hydroxyethyl)-Adenosine Exhibits Insecticidal Activity against Plutella xylostella via Adenosine Receptors

    Fang, Ming; Chai, Yiqiu; Chen, Guanjv; Wang, Huidong; Huang, Bo


    The diamondback moth, Plutella xylostella, is one of the most important pests of cruciferous crops. We have earlier shown that N6-(2-hydroxyethyl)-adenosine (HEA) exhibits insecticidal activity against P. xylostella. In the present study we investigated the possible mechanism of insecticidal action of HEA on P. xylostella. HEA is a derivative of adenosine, therefore, we speculated whether it acts via P. xylostella adenosine receptor (PxAdoR). We used RNAi approach to silence PxAdoR gene and used antagonist of denosine receptor (AdoR) to study the insecticidal effect of HEA. We cloned the whole sequence of PxAdoR gene. A BLAST search using NCBI protein database showed a 61% identity with the Drosophila adenosine receptor (DmAdoR) and a 32–35% identity with human AdoR. Though the amino acids sequence of PxAdoR was different compared to other adenosine receptors, most of the amino acids that are known to be important for adenosine receptor ligand binding and signaling were present. However, only 30% binding sites key residues was similar between PxAdoR and A1R. HEA, at a dose of 1 mg/mL, was found to be lethal to the second-instar larvae of P. xylostella, and a significant reduction of mortality and growth inhibition ratio were obtained when HEA was administered to the larvae along with PxAdoR-dsRNA or antagonist of AdoR (SCH58261) for 36, 48, or 60 h. Especially at 48 h, the rate of growth inhibition of the PxAdoR knockdown group was 3.5-fold less than that of the HEA group, and the corrected mortality of SCH58261 group was reduced almost 2-fold compared with the HEA group. Our findings show that HEA may exert its insecticidal activity against P. xylostella larvae via acting on PxAdoR. PMID:27668428

  5. N6-(2-Hydroxyethyl)-Adenosine Exhibits Insecticidal Activity against Plutella xylostella via Adenosine Receptors.

    Fang, Ming; Chai, Yiqiu; Chen, Guanjv; Wang, Huidong; Huang, Bo

    The diamondback moth, Plutella xylostella, is one of the most important pests of cruciferous crops. We have earlier shown that N6-(2-hydroxyethyl)-adenosine (HEA) exhibits insecticidal activity against P. xylostella. In the present study we investigated the possible mechanism of insecticidal action of HEA on P. xylostella. HEA is a derivative of adenosine, therefore, we speculated whether it acts via P. xylostella adenosine receptor (PxAdoR). We used RNAi approach to silence PxAdoR gene and used antagonist of denosine receptor (AdoR) to study the insecticidal effect of HEA. We cloned the whole sequence of PxAdoR gene. A BLAST search using NCBI protein database showed a 61% identity with the Drosophila adenosine receptor (DmAdoR) and a 32-35% identity with human AdoR. Though the amino acids sequence of PxAdoR was different compared to other adenosine receptors, most of the amino acids that are known to be important for adenosine receptor ligand binding and signaling were present. However, only 30% binding sites key residues was similar between PxAdoR and A1R. HEA, at a dose of 1 mg/mL, was found to be lethal to the second-instar larvae of P. xylostella, and a significant reduction of mortality and growth inhibition ratio were obtained when HEA was administered to the larvae along with PxAdoR-dsRNA or antagonist of AdoR (SCH58261) for 36, 48, or 60 h. Especially at 48 h, the rate of growth inhibition of the PxAdoR knockdown group was 3.5-fold less than that of the HEA group, and the corrected mortality of SCH58261 group was reduced almost 2-fold compared with the HEA group. Our findings show that HEA may exert its insecticidal activity against P. xylostella larvae via acting on PxAdoR.

  6. Ribonucleic acid stimulation of mammalian liver nuclear-envelope nucleoside triphosphatase. A possible enzymic marker for the nuclear envelope.

    Agutter, P S; Harris, J R; Stevenson, I


    1. The specific activity of rat and pig liver nuclear-envelope nucleoside triphosphatase (EC decreases when the system is depleted of RNA. The activity can be restored by adding high concentrations of yeast RNA to the assay medium. 2. Exogenous RNA also increases the activity of the enzyme in control envelopes (not RNA-depleted). The effect appears to be largely specific for poly(A) and poly(G); it is not stimulated by rRNA or tRNA preparations, ribonuclease-hydrolysed RNA, AMP, or double- or single-stranded DNA. 3. Inhibitors of the enzyme, in concentrations at which half-maximal inhibition of the enzyme is achieved, do not affect the percentage stimulation of the enzyme by yeast RNA. 4. The simulation is abolished by the inclusion of 150 mM-KCl or -NaCl in the assay medium, but not by increasing the assay pH to 8.5. 5. The results are discussed in the light of the possible role of the nucleoside triphosphatase in vivo in nucleo-cytoplasmic ribonucleoprotein translocation. 6. It is proposed that poly(G)-stimulated Mg2+-activated adenosine triphosphatase activity should be adopted as an enzymic marker for the nuclear envelope.

  7. Different efficacy of adenosine and NECA derivatives at the human A3 adenosine receptor: insight into the receptor activation switch.

    Dal Ben, Diego; Buccioni, Michela; Lambertucci, Catia; Kachler, Sonja; Falgner, Nico; Marucci, Gabriella; Thomas, Ajiroghene; Cristalli, Gloria; Volpini, Rosaria; Klotz, Karl-Norbert


    A3 Adenosine receptors are promising drug targets for a number of diseases and intense efforts are dedicated to develop selective agonists and antagonists of these receptors. A series of adenosine derivatives with 2-(ar)-alkynyl chains, with high affinity and different degrees of selectivity for human A3 adenosine receptors was tested for the ability to inhibit forskolin-stimulated adenylyl cyclase. All these derivatives are partial agonists at A3 adenosine receptors; their efficacy is not significantly modified by the introduction of small alkyl substituents in the N(6)-position. In contrast, the adenosine-5'-N-ethyluronamide (NECA) analogs of 2-(ar)-alkynyladenosine derivatives are full A3 agonists. Molecular modeling analyses were performed considering both the conformational behavior of the ligands and the impact of 2- and 5'-substituents on ligand-target interaction. The results suggest an explanation for the different agonistic behavior of adenosine and NECA derivatives, respectively. A sub-pocket of the binding site was analyzed as a crucial interaction domain for receptor activation.

  8. Ribosome-inactivating lectins with polynucleotide:adenosine glycosidase activity.

    Battelli, M G; Barbieri, L; Bolognesi, A; Buonamici, L; Valbonesi, P; Polito, L; Van Damme, E J; Peumans, W J; Stirpe, F


    Lectins from Aegopodium podagraria (APA), Bryonia dioica (BDA), Galanthus nivalis (GNA), Iris hybrid (IRA) and Sambucus nigra (SNAI), and a new lectin-related protein from Sambucus nigra (SNLRP) were studied to ascertain whether they had the properties of ribosome-inactivating proteins (RIP). IRA and SNLRP inhibited protein synthesis by a cell-free system and, at much higher concentrations, by cells and had polynucleotide:adenosine glycosidase activity, thus behaving like non-toxic type 2 (two chain) RIP. APA and SNAI had much less activity, and BDA and GNA did not inhibit protein synthesis.

  9. The Rickettsia prowazekii invasion gene homolog (invA) encodes a Nudix hydrolase active on adenosine (5')-pentaphospho-(5')-adenosine.

    Gaywee, Jariyanart; Xu, WenLian; Radulovic, Suzana; Bessman, Maurice J; Azad, Abdu F


    The genomic sequence of Rickettsia prowazekii, the obligate intracellular bacterium responsible for epidemic typhus, reveals an uncharacterized invasion gene homolog (invA). The deduced protein of 18,752 Da contains a Nudix signature, the specific motif found in the Nudix hydrolase family. To characterize the function of InvA, the gene was cloned and overexpressed in Escherichia coli. The expressed protein was purified to near homogeneity and subsequently tested for its enzymatic activity against a series of nucleoside diphosphate derivatives. The purified InvA exhibits hydrolytic activity toward dinucleoside oligophosphates (Np(n)N; n > or = 5), a group of cellular signaling molecules. At optimal pH 8.5, the enzyme actively degrades adenosine (5')-pentaphospho-(5')-adenosine into ATP and ADP with a K(m) of 0.1 mM and k(cat) of 1.9 s(-1). Guanosine (5')-pentaphospho-(5')-guanosine and adenosine-(5')-hexaphospho (5')-adenosine are also substrates. Similar to other Nudix hydrolases, InvA requires a divalent metal cation, Mg(2+) or Zn(2+), for optimal activity. These data suggest that the rickettsial invasion protein likely plays a role in controlling the concentration of stress-induced dinucleoside oligophosphates following bacterial invasion.

  10. Methylthioadenosine reprograms macrophage activation through adenosine receptor stimulation.

    Peter A Keyel

    Full Text Available Regulation of inflammation is necessary to balance sufficient pathogen clearance with excessive tissue damage. Central to regulating inflammation is the switch from a pro-inflammatory pathway to an anti-inflammatory pathway. Macrophages are well-positioned to initiate this switch, and as such are the target of multiple therapeutics. One such potential therapeutic is methylthioadenosine (MTA, which inhibits TNFα production following LPS stimulation. We found that MTA could block TNFα production by multiple TLR ligands. Further, it prevented surface expression of CD69 and CD86 and reduced NF-KB signaling. We then determined that the mechanism of this action by MTA is signaling through adenosine A2 receptors. A2 receptors and TLR receptors synergized to promote an anti-inflammatory phenotype, as MTA enhanced LPS tolerance. In contrast, IL-1β production and processing was not affected by MTA exposure. Taken together, these data demonstrate that MTA reprograms TLR activation pathways via adenosine receptors to promote resolution of inflammation.

  11. The expression of copper-transporting P-type adenosine triphosphatase in ovarian cancer and its correlation with chemotherapy resistance%P型铜转运ATP酶在卵巢癌中的表达及其与化疗耐药的关系

    李侠; 吴绪峰; 陈惠祯


    目的:探讨P型铜转运ATP酶(copper-transporting P-type adenosine triphosphatase,ATP 7B)在卵巢癌组织中的表达及其与临床病理参数、化疗耐药的关系.方法:用半定量RT-PCR技术检测ATP 7B在36例卵巢癌组织,10例良性卵巢肿瘤组织,10例正常卵巢组织中的表达水平.结果:ATP7B在卵巢癌组织中的阳性表达率为38.9%,显著高于在良性卵巢肿瘤(0%)及正常卵巢组织中(0%)的表达(P<0.01).ATP7B的表达与肿瘤分化程度有关,ATP7B在低分化组织中的表达明显高于高、中分化组(P<0.05).ATP7B在术前化疗组中的表达高于术前未化疗组,差异有统计学意义(P<0.05).ATP7B表达阳性的卵巢癌患者对化疗的反应率(28.6%)明显低于表达为阴性的患者(72.7%)(P<0.05).结论:ATP 7B可能在卵巢癌对铂类抗肿瘤药的耐药过程中发挥了重要作用.

  12. Effects of vaccinia virus uracil DNA glycosylase catalytic site and deoxyuridine triphosphatase deletion mutations individually and together on replication in active and quiescent cells and pathogenesis in mice

    Moss Bernard


    Full Text Available Abstract Background Low levels of uracil in DNA result from misincorporation of dUMP or cytosine deamination. Vaccinia virus (VACV, the prototype poxvirus, encodes two enzymes that can potentially reduce the amount of uracil in DNA. Deoxyuridine triphosphatase (dUTPase hydrolyzes dUTP, generating dUMP for biosynthesis of thymidine nucleotides while decreasing the availability of dUTP for misincorporation; uracil DNA glycosylase (UNG cleaves uracil N-glycosylic bonds in DNA initiating base excision repair. Studies with actively dividing cells showed that the VACV UNG protein is required for DNA replication but the UNG catalytic site is not, whereas the dUTPase gene can be deleted without impairing virus replication. Recombinant VACV with an UNG catalytic site mutation was attenuated in vivo, while a dUTPase deletion mutant was not. However, the importance of the two enzymes for replication in quiescent cells, their possible synergy and roles in virulence have not been fully assessed. Results VACV mutants lacking the gene encoding dUTPase or with catalytic site mutations in UNG and double UNG/dUTPase mutants were constructed. Replication of UNG and UNG/dUTPase mutants were slightly reduced compared to wild type or the dUTPase mutant in actively dividing cells. Viral DNA replication was reduced about one-third under these conditions. After high multiplicity infection of quiescent fibroblasts, yields of wild type and mutant viruses were decreased by 2-logs with relative differences similar to those observed in active fibroblasts. However, under low multiplicity multi-step growth conditions in quiescent fibroblasts, replication of the dUTPase/UNG mutant was delayed and 5-fold lower than that of either single mutant or parental virus. This difference was exacerbated by 1-day serial passages on quiescent fibroblasts, resulting in 2- to 3-logs lower titer of the double mutant compared to the parental and single mutant viruses. Each mutant was more

  13. Adenosine activates brown adipose tissue and recruits beige adipocytes via A2A receptors

    Gnad, Thorsten; Scheibler, Saskia; von Kügelgen, Ivar


    hamster or rat. However, the role of adenosine in human BAT is unknown. Here we show that adenosine activates human and murine brown adipocytes at low nanomolar concentrations. Adenosine is released in BAT during stimulation of sympathetic nerves as well as from brown adipocytes. The adenosine A2A...... of A2A receptors or injection of lentiviral vectors expressing the A2A receptor into white fat induces brown-like cells-so-called beige adipocytes. Importantly, mice fed a high-fat diet and treated with an A2A agonist are leaner with improved glucose tolerance. Taken together, our results demonstrate...... that adenosine-A2A signalling plays an unexpected physiological role in sympathetic BAT activation and protects mice from diet-induced obesity. Those findings reveal new possibilities for developing novel obesity therapies....




    Full Text Available Meningitis is inflammation of the meninges (pia, arachnoid and dura mater covering the brain and the spinal cord. ADA is an enzyme in the purine salvage pathway which is found in abundance in active T-lymphocytes. Hence, an attempt was made to estimate the CSF ADA level in patients with suspected meningitis and throw light on its use in differentiating the various types of meningitis. AIMS AND OBJECTIVES To estimate the level of CSF adenosine deaminase level in different types of meningitis. To assess its usefulness in differentiating the various types (bacterial, viral and tuberculous of meningitis. MATERIALS AND METHODS The study was conducted at the medical wards of Govt. Rajaji Hospital, Madurai, a prospective analytical study from a period of April 2012 to September 2012. OBSERVATION AND RESULTS Tuberculous meningitis occurred more in the age group of 21–40 years. Bacterial meningitis was seen mainly in patients < 20 years of age. Viral meningitis was seen in all age groups. CSF ADA level was highest in tuberculous meningitis, the mean value being 24.5 U/L. The mean value of ADA in bacterial meningitis was 4.54 U/L and viral meningitis patients had lowest mean ADA value of 2.65 U/L. CONCLUSION In our study, 50 patients with meningitis admitted in Government Rajaji Hospital from April 2012 to September 2012 were evaluated. Meningitis predominantly affected people in the age group of 20-40 years in our study with a male: female ratio of 1.9:1. Cases of tuberculous meningitis constituted 48% of the study group and bacterial and viral meningitis were 26% each. CSF protein values were higher and sugar values lower in patients with tuberculous and bacterial meningitis. CSF cell counts were higher in patients with bacterial meningitis.

  15. Adenosine A3 receptor activation is neuroprotective against retinal neurodegeneration.

    Galvao, Joana; Elvas, Filipe; Martins, Tiago; Cordeiro, M Francesca; Ambrósio, António Francisco; Santiago, Ana Raquel


    Death of retinal neural cells, namely retinal ganglion cells (RGCs), is a characteristic of several retinal neurodegenerative diseases. Although the role of adenosine A3 receptor (A3R) in neuroprotection is controversial, A3R activation has been reported to afford protection against several brain insults, with few studies in the retina. In vitro models (retinal neural and organotypic cultures) and animal models [ischemia-reperfusion (I-R) and partial optic nerve transection (pONT)] were used to study the neuroprotective properties of A3R activation against retinal neurodegeneration. The A3R selective agonist (2-Cl-IB-MECA, 1 μM) prevented apoptosis (TUNEL(+)-cells) induced by kainate and cyclothiazide (KA + CTZ) in retinal neural cultures (86.5 ± 7.4 and 37.2 ± 6.1 TUNEL(+)-cells/field, in KA + CTZ and KA + CTZ + 2-Cl-IB-MECA, respectively). In retinal organotypic cultures, 2-Cl-IB-MECA attenuated NMDA-induced cell death, assessed by TUNEL (17.3 ± 2.3 and 8.3 ± 1.2 TUNEL(+)-cells/mm(2) in NMDA and NMDA+2-Cl-IB-MECA, respectively) and PI incorporation (ratio DIV4/DIV2 3.3 ± 0.3 and 1.3 ± 0.1 in NMDA and NMDA+2-Cl-IB-MECA, respectively) assays. Intravitreal 2-Cl-IB-MECA administration afforded protection against I-R injury decreasing the number of TUNEL(+) cells by 72%, and increased RGC survival by 57%. Also, intravitreal administration of 2-Cl-IB-MECA inhibited apoptosis (from 449.4 ± 37.8 to 207.6 ± 48.9 annexin-V(+)-cells) and RGC loss (from 1.2 ± 0.6 to 8.1 ± 1.7 cells/mm) induced by pONT. This study demonstrates that 2-Cl-IB-MECA is neuroprotective to the retina, both in vitro and in vivo. Activation of A3R may have great potential in the management of retinal neurodegenerative diseases characterized by RGC death, as glaucoma and diabetic retinopathy, and ischemic diseases.

  16. Influence of nucleotides, cations and nucleoside triphosphatase inhibitors on the release of ribonucleic acid from isolated rat liver nuclei.

    Agutter, P S


    The reasons underlying reported discrepancies in the effects of ATP, ADP, adenosine 5'-[beta gamma-methylene]triphosphate, AMP + PPi, P-chloromercuribenzoate and F- on RNA efflux from isolated rat liver nuclei and on nuclear envelope nucleoside triphosphatase activity were investigated. The stimulatory effect of ADP was attributed to myokinase activity associated with the nuclei; this activity was eluted on repeated washing with nuclear incubation medium. In the absence of Ca2+ and Mn2+, ATP, adenosine 5'[beta gamma-methylene]triphosphate and AMP +PPi were found to promote release of both DNA and RNA. In the presence of 0.5 mM-Ca2+ and 9.3 mM-Mn2+, only ATP promoted RNA efflux to a significant extent. In the absence of spermidine, Ca2+ and Mn2+, nuclei released large quantities of DNA and RNA into the medium; this effect was promoted by p-chloromereuribenzoate. In the presence of the three cations, however, p-chloromercuribenzoate inhibited RNA efflux. F- caused a slight leakage of DNA from nuclei. The results are discussed in terms of models for the effects of ATP and analogues on RNA efflux and nuclear stability.

  17. Suppression of adenosine-activated chloride transport by ethanol in airway epithelia.

    Sammeta V Raju

    Full Text Available Alcohol abuse is associated with increased lung infections. Molecular understanding of the underlying mechanisms is not complete. Airway epithelial ion transport regulates the homeostasis of airway surface liquid, essential for airway mucosal immunity and lung host defense. Here, air-liquid interface cultures of Calu-3 epithelial cells were basolaterally exposed to physiologically relevant concentrations of ethanol (0, 25, 50 and 100 mM for 24 hours and adenosine-stimulated ion transport was measured by Ussing chamber. The ethanol exposure reduced the epithelial short-circuit currents (I(SC in a dose-dependent manner. The ion currents activated by adenosine were chloride conductance mediated by cystic fibrosis transmembrane conductance regulator (CFTR, a cAMP-activated chloride channel. Alloxazine, a specific inhibitor for A(2B adenosine receptor (A(2BAR, largely abolished the adenosine-stimulated chloride transport, suggesting that A(2BAR is a major receptor responsible for regulating the chloride transport of the cells. Ethanol significantly reduced intracellular cAMP production upon adenosine stimulation. Moreover, ethanol-suppression of the chloride secretion was able to be restored by cAMP analogs or by inhibitors to block cAMP degradation. These results imply that ethanol exposure dysregulates CFTR-mediated chloride transport in airways by suppression of adenosine-A(2BAR-cAMP signaling pathway, which might contribute to alcohol-associated lung infections.

  18. Protective effects of inhibition of adenosine monophosphate activated protein kinase activity against cerebral ischemia-reperfusion injury in mice



    Objective To observe the effect of inhibition of adenosine monophosphate activated protein kinase (AMPK) on shape,function and inflammatory factor of microglia for mice after cerebral ischemia-reperfusion

  19. Activation of NTS A(1) adenosine receptors inhibits regional sympathetic responses evoked by activation of cardiopulmonary chemoreflex.

    Ichinose, Tomoko K; Minic, Zeljka; Li, Cailian; O'Leary, Donal S; Scislo, Tadeusz J


    Previously we have shown that adenosine operating via the A(1) receptor subtype may inhibit glutamatergic transmission in the baroreflex arc within the nucleus of the solitary tract (NTS) and differentially increase renal (RSNA), preganglionic adrenal (pre-ASNA), and lumbar (LSNA) sympathetic nerve activity (ASNA>RSNA≥LSNA). Since the cardiopulmonary chemoreflex and the arterial baroreflex are mediated via similar medullary pathways, and glutamate is a primary transmitter in both pathways, it is likely that adenosine operating via A(1) receptors in the NTS may differentially inhibit regional sympathetic responses evoked by activation of cardiopulmonary chemoreceptors. Therefore, in urethane-chloralose-anesthetized rats (n = 37) we compared regional sympathoinhibition evoked by the cardiopulmonary chemoreflex (activated with right atrial injections of serotonin 5HT(3) receptor agonist phenylbiguanide, PBG, 1-8 μg/kg) before and after selective stimulation of NTS A(1) adenosine receptors [microinjections of N(6)-cyclopentyl adenosine (CPA), 0.033-330 pmol/50 nl]. Activation of cardiopulmonary chemoreceptors evoked differential, dose-dependent sympathoinhibition (RSNA>ASNA>LSNA), and decreases in arterial pressure and heart rate. These differential sympathetic responses were uniformly attenuated in dose-dependent manner by microinjections of CPA into the NTS. Volume control (n = 11) and blockade of adenosine receptor subtypes in the NTS via 8-(p-sulfophenyl)theophylline (8-SPT, 1 nmol in 100 nl) (n = 9) did not affect the reflex responses. We conclude that activation of NTS A(1) adenosine receptors uniformly inhibits neural and cardiovascular cardiopulmonary chemoreflex responses. A(1) adenosine receptors have no tonic modulatory effect on this reflex under normal conditions. However, when adenosine is released into the NTS (i.e., during stress or severe hypotension/ischemia), it may serve as negative feedback regulator for depressor and sympathoinhibitory reflexes

  20. Nanomolar Inhibitors of Trypanosoma brucei RNA Triphosphatase

    Paul Smith


    Full Text Available Eukaryal taxa differ with respect to the structure and mechanism of the RNA triphosphatase (RTPase component of the mRNA capping apparatus. Protozoa, fungi, and certain DNA viruses have a metal-dependent RTPase that belongs to the triphosphate tunnel metalloenzyme (TTM superfamily. Because the structures, active sites, and chemical mechanisms of the TTM-type RTPases differ from those of mammalian RTPases, the TTM RTPases are potential targets for antiprotozoal, antifungal, and antiviral drug discovery. Here, we employed RNA interference (RNAi knockdown methods to show that Trypanosoma brucei RTPase Cet1 (TbCet1 is necessary for proliferation of procyclic cells in culture. We then conducted a high-throughput biochemical screen for small-molecule inhibitors of the phosphohydrolase activity of TbCet1. We identified several classes of chemicals—including chlorogenic acids, phenolic glycopyranosides, flavonoids, and other phenolics—that inhibit TbCet1 with nanomolar to low-micromolar 50% inhibitory concentrations (IC50s. We confirmed the activity of these compounds, and tested various analogs thereof, by direct manual assays of TbCet1 phosphohydrolase activity. The most potent nanomolar inhibitors included tetracaffeoylquinic acid, 5-galloylgalloylquinic acid, pentagalloylglucose, rosmarinic acid, and miquelianin. TbCet1 inhibitors were less active (or inactive against the orthologous TTM-type RTPases of mimivirus, baculovirus, and budding yeast (Saccharomyces cerevisiae. Our results affirm that a TTM RTPase is subject to potent inhibition by small molecules, with the caveat that parallel screens against TTM RTPases from multiple different pathogens may be required to fully probe the chemical space of TTM inhibition.




    In experiment A, the activity of the glomerular antithrombotic enzyme adenosine diphosphatase (ADPase) and the sensitivity of this enzyme for endotoxin (1.0 mug/kg BW) in various reproductive conditions of female rats were studied through use of enzyme histochemical methods. In experiment B, the eff

  2. Adenosine inhibits neutrophil vascular endothelial growth factor release and transendothelial migration via A2B receptor activation.

    Wakai, A


    The effects of adenosine on neutrophil (polymorphonuclear neutrophils; PMN)-directed changes in vascular permeability are poorly characterized. This study investigated whether adenosine modulates activated PMN vascular endothelial growth factor (vascular permeability factor; VEGF) release and transendothelial migration. PMN activated with tumour necrosis factor-alpha (TNF-alpha, 10 ng\\/mL) were incubated with adenosine and its receptor-specific analogues. Culture supernatants were assayed for VEGF. PMN transendothelial migration across human umbilical vein endothelial cell (HUVEC) monolayers was assessed in vitro. Adhesion molecule receptor expression was assessed flow cytometrically. Adenosine and some of its receptor-specific analogues dose-dependently inhibited activated PMN VEGF release. The rank order of potency was consistent with the affinity profile of human A2B receptors. The inhibitory effect of adenosine was reversed by 3,7-dimethyl-1-propargylxanthine, an A2 receptor antagonist. Adenosine (100 microM) or the A2B receptor agonist 5\\'-N-ethylcarboxamidoadenosine (NECA, 100 microM) significantly reduced PMN transendothelial migration. However, expression of activated PMN beta2 integrins and HUVEC ICAM-1 were not significantly altered by adenosine or NECA. Adenosine attenuates human PMN VEGF release and transendothelial migration via the A2B receptor. This provides a novel target for the modulation of PMN-directed vascular hyperpermeability in conditions such as the capillary leak syndrome.

  3. Adenosine Monophosphate (AMP)-Activated Protein Kinase: A New Target for Nutraceutical Compounds.

    Marín-Aguilar, Fabiola; Pavillard, Luis E; Giampieri, Francesca; Bullón, Pedro; Cordero, Mario D


    Adenosine monophosphate-activated protein kinase (AMPK) is an important energy sensor which is activated by increases in adenosine monophosphate (AMP)/adenosine triphosphate (ATP) ratio and/or adenosine diphosphate (ADP)/ATP ratio, and increases different metabolic pathways such as fatty acid oxidation, glucose transport and mitochondrial biogenesis. In this sense, AMPK maintains cellular energy homeostasis by induction of catabolism and inhibition of ATP-consuming biosynthetic pathways to preserve ATP levels. Several studies indicate a reduction of AMPK sensitivity to cellular stress during aging and this could impair the downstream signaling and the maintenance of the cellular energy balance and the stress resistance. However, several diseases have been related with an AMPK dysfunction. Alterations in AMPK signaling decrease mitochondrial biogenesis, increase cellular stress and induce inflammation, which are typical events of the aging process and have been associated to several pathological processes. In this sense, in the last few years AMPK has been identified as a very interesting target and different nutraceutical compounds are being studied for an interesting potential effect on AMPK induction. In this review, we will evaluate the interaction of the different nutraceutical compounds to induce the AMPK phosphorylation and the applications in diseases such as cancer, type II diabetes, neurodegenerative diseases or cardiovascular diseases.

  4. Pyrazolo Derivatives as Potent Adenosine Receptor Antagonists: An Overview on the Structure-Activity Relationships

    Siew Lee Cheong


    Full Text Available In the past few decades, medicinal chemistry research towards potent and selective antagonists of human adenosine receptors (namely, A1, A2A, A2B, and A3 has been evolving rapidly. These antagonists are deemed therapeutically beneficial in several pathological conditions including neurological and renal disorders, cancer, inflammation, and glaucoma. Up to this point, many classes of compounds have been successfully synthesized and identified as potent human adenosine receptor antagonists. In this paper, an overview of the structure-activity relationship (SAR profiles of promising nonxanthine pyrazolo derivatives is reported and discussed. We have emphasized the SAR for some representative structures such as pyrazolo-[4,3-e]-1,2,4-triazolo-[1,5-c]pyrimidines; pyrazolo-[3,4-c] or -[4,3-c]quinolines; pyrazolo-[4,3-d]pyrimidinones; pyrazolo-[3,4-d]pyrimidines and pyrazolo-[1,5-a]pyridines. This overview not only clarifies the structural requirements deemed essential for affinity towards individual adenosine receptor subtypes, but it also sheds light on the rational design and optimization of existing structural templates to allow us to conceive new, more potent adenosine receptor antagonists.

  5. Platelet aggregation and serum adenosine deaminase (ADA) activity in pregnancy associated with diabetes, hypertension and HIV.

    Leal, Claudio A M; Leal, Daniela B R; Adefegha, Stephen A; Morsch, Vera M; da Silva, José E P; Rezer, João F P; Schrekker, Clarissa M L; Abdalla, Faida H; Schetinger, Maria R C


    Platelet aggregation and adenosine deaminase (ADA) activity were evaluated in pregnant women living with some disease conditions including hypertension, diabetes mellitus and human immunodeficiency virus infection. The subject population is consisted of 15 non-pregnant healthy women [control group (CG)], 15 women with normal pregnancy (NP), 7 women with hypertensive pregnancy (HP), 10 women with gestational diabetes mellitus (GDM) and 12 women with human immunodeficiency virus-infected pregnancy (HIP) groups. The aggregation of platelets was checked using an optical aggregometer, and serum ADA activity was determined using the colorimetric method. After the addition of 5 µM of agonist adenosine diphosphate, the percentage of platelet aggregation was significantly (p pregnancy and pregnancy-associated diseases suggest that platelet aggregation and ADA activity could serve as peripheral markers for the development of effective therapy in the maintenance of homeostasis and some inflammatory process in these pathophysiological conditions. Copyright © 2016 John Wiley & Sons, Ltd.

  6. Repetitive systemic morphine alters activity-dependent plasticity of Schaffer-collateral-CA1 pyramidal cell synapses: involvement of adenosine A1 receptors and adenosine deaminase.

    Sadegh, Mehdi; Fathollahi, Yaghoub


    The effectiveness of O-pulse stimulation (TPS) for the reversal of O-pattern primed bursts (PB)-induced long-term potentiation (LTP) were examined at the Schaffer-collateral-CA1 pyramidal cell synapses of hippocampal slices derived from rats chronically treated with morphine (M-T). The results showed that slices derived from both control and M-T rats had normal field excitatory postsynaptic potential (fEPSP)-LTP, whereas PS-LTP in slices from M-T rats was significantly greater than that from control slices. When morphine was applied in vitro to slices derived from rats chronically treated with morphine, the augmentation of PS-LTP was not seen. TPS given 30 min after LTP induction failed to reverse the fEPSP- or PS-LTP in both groups of slices. However, TPS delivered in the presence of long-term in vitro morphine caused the PS-LTP reversal. This effect was blocked by the adenosine A1 receptor (A1R) antagonist CPX (200 nM) and furthermore was enhanced by the adenosine deaminase (ADA) inhibitor EHNA (10 μM). Interestingly, TPS given 30 min after LTP induction in the presence of EHNA (10 μM) can reverse LTP in morphine-exposed control slices in vitro. These results suggest adaptive changes in the hippocampus area CA1 in particular in adenosine system following repetitive systemic morphine. Chronic in vivo morphine increases A1R and reduces ADA activity in the hippocampus. Consequently, adenosine can accumulate because of a stimulus train-induced activity pattern in CA1 area and takes the opportunity to work as an inhibitory neuromodulator and also to enable CA1 to cope with chronic morphine. In addition, adaptive mechanisms are differentially working in the dendrite layer rather than the somatic layer of hippocampal CA1.

  7. Activation of NTS A2a adenosine receptors differentially resets baroreflex control of renal vs. adrenal sympathetic nerve activity.

    Ichinose, Tomoko K; O'Leary, Donal S; Scislo, Tadeusz J


    The role of nucleus of solitary tract (NTS) A(2a) adenosine receptors in baroreflex mechanisms is controversial. Stimulation of these receptors releases glutamate within the NTS and elicits baroreflex-like decreases in mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA), whereas inhibition of these receptors attenuates HR baroreflex responses. In contrast, stimulation of NTS A(2a) adenosine receptors increases preganglionic adrenal sympathetic nerve activity (pre-ASNA), and the depressor and sympathoinhibitory responses are not markedly affected by sinoaortic denervation and blockade of NTS glutamatergic transmission. To elucidate the role of NTS A(2a) adenosine receptors in baroreflex function, we compared full baroreflex stimulus-response curves for HR, RSNA, and pre-ASNA (intravenous nitroprusside/phenylephrine) before and after bilateral NTS microinjections of selective adenosine A(2a) receptor agonist (CGS-21680; 2.0, 20 pmol/50 nl), selective A(2a) receptor antagonist (ZM-241385; 40 pmol/100 nl), and nonselective A(1) + A(2a) receptor antagonist (8-SPT; 1 nmol/100 nl) in urethane/alpha-chloralose anesthetized rats. Activation of A(2a) receptors decreased the range, upper plateau, and gain of baroreflex-response curves for RSNA, whereas these parameters all increased for pre-ASNA, consistent with direct effects of the agonist on regional sympathetic activity. However, no resetting of baroreflex-response curves along the MAP axis occurred despite the marked decreases in baseline MAP. The antagonists had no marked effects on baseline variables or baroreflex-response functions. We conclude that the activation of NTS A(2a) adenosine receptors differentially alters baroreflex control of HR, RSNA, and pre-ASNA mostly via non-baroreflex mechanism(s), and these receptors have virtually no tonic action on baroreflex control of these sympathetic outputs.

  8. Adenosine and adenosine receptors: Newer therapeutic perspective

    Manjunath S


    Full Text Available Adenosine, a purine nucleoside has been described as a ′retaliatory metabolite′ by virtue of its ability to function in an autocrine manner and to modify the activity of a range of cell types, following its extracellular accumulation during cell stress or injury. These effects are largely protective and are triggered by binding of adenosine to any of the four adenosine receptor subtypes namely A1, A2a, A2b, A3, which have been cloned in humans, and are expressed in most of the organs. Each is encoded by a separate gene and has different functions, although overlapping. For instance, both A1 and A2a receptors play a role in regulating myocardial oxygen consumption and coronary blood flow. It is a proven fact that adenosine plays pivotal role in different physiological functions, such as induction of sleep, neuroprotection and protection against oxidative stress. Until now adenosine was used for certain conditions like paroxysmal supraventricular tachycardia (PSVT and Wolff Parkinson White (WPW syndrome. Now there is a growing evidence that adenosine receptors could be promising therapeutic targets in a wide range of conditions including cardiac, pulmonary, immunological and inflammatory disorders. After more than three decades of research in medicinal chemistry, a number of selective agonists and antagonists of adenosine receptors have been discovered and some have been clinically evaluated, although none has yet received regulatory approval. So this review focuses mainly on the newer potential role of adenosine and its receptors in different clinical conditions.

  9. Endogenous activation of adenosine A1 receptors promotes post-ischemic electrocortical burst suppression

    Ilie, A; Ciocan, D; Constantinescu, A O


    . Several lines of evidence suggest that BS reflects an impairment of neocortical connectivity. Here we tested in vivo whether synaptic depression by adenosine A1 receptor (A1R) activation contributes to BS patterns following GCI. Male Wistar rats were subjected to 1, 5 or 10 min of GCI using a "four...... of post-ischemic BS patterns following brief ischemic episodes. It is likely that synaptic depression by post-ischemic A1R activation functionally disrupts the connectivity within the cortical networks to an extent that promotes BS patterns....

  10. Beneficial effects of metformin on primary cardiomyocytes via activation of adenosine monophosphate-activated protein kinase

    WANG Xiao-fang; ZHANG Jin-ying; LI Ling; ZHAO Xiao-yan


    Background Metformin has become a cornerstone in the treatment of patients with type-2 diabetes. Accumulated evidence suggests that metformin supports direct cardiovascular effects. The present study aimed to investigate if metformin has beneficial effects on primary cardiomyocytes damaged by H2O2, and reveal the potential mechanism of action of metformin.Methods Cardiomyocytes were incubated in the presence of 100 umol/L. H2O2 for 12 hours. Cardiomyocytes were pretreated with metformin at different concentrations and time and with aminoimidazole carboxamide ribonucleotide (AICAR) (500 umol/L), an adenosine monophophate (AMP)-activated protein kinase (AMPK) agonist for 60 minutes before the addition of H2O2. Other cells were preincubated with compound C (an AMPK antagonist, 20 umol/L) for 4 hours. The viability and apoptosis of cells were analyzed. AMPK, endothelial nitric oxide synthase (eNOS), and transforming growth factor (TGF)-β1 were analyzed using immunblotting.Results Metformin had antagonistic effects on the influences of H2O2 on cell viability and attenuated oxidative stress-induced apoptosis. Metformin also increased phosphorylation of AMPK and eNOS, and reduced the expression of TGF-β1, basic fibroblast growth factor (bFGF), and tumor necrosis factor (TNF)-α.Conclusions Metformin has beneficial effects on cardiomyocytes, and this effect involves activation of the AMPK-eNOS pathway. Metformin may be potentially beneficial for the treatment of heart disease.

  11. Adenosine deaminase activity in serum and lymphocytes of rats infected with Sporothrix schenckii.

    Castro, Verônica S P; Pimentel, Victor C; Da Silva, Aleksandro S; Thomé, Gustavo R; Wolkmer, Patrícia; Castro, Jorge L C; Costa, Márcio M; da Silva, Cássia B; Oliveira, Daniele C; Alves, Sydney H; Schetinger, Maria R C; Lopes, Sonia T A; Mazzanti, Cinthia M


    Sporotrichosis is a fungal infection of subcutaneous or chronic evolution, inflammatory lesions characterized by their pyogranulomatous aspect, caused by the dimorphic fungus Sporothrix schenckii. Adenosine deaminase (ADA) is a "key" enzyme in the purine metabolism, promoting the deamination of adenosine, an important anti-inflammatory molecule. The increase in ADA activity has been demonstrated in several inflammatory conditions; however, there are no data in the literature associated with this fungal infection. The objective of this study was to evaluate the activity of serum ADA (S-ADA) and lymphocytes (L-ADA) of rats infected with S. schenckii. We used seventy-eight rats divided into two groups. In the first experiment, rats were infected subcutaneously and in the second experiment, infected intraperitoneally. Blood samples for hematologic evaluation and activities of S-ADA and L-ADA were performed at days 15, 30, and 40 post-infection (PI) to assess disease progression. In the second experiment, it was observed an acute decrease in activity of S-ADA and L-ADA (P schenckii alters the activities of S-ADA in experimentally infected rats, demonstrating the involvement of this enzyme in the pathogenesis of sporotrichosis.

  12. Activation and modulation of cardiac poly-adenosine diphosphate ribose polymerase activity in a rat model of brain death.

    Brain, John G; Rostron, Anthony J; Dark, John H; Kirby, John A


    DNA damage during transplantation can activate poly-adenosine diphosphate ribose polymerase (PARP) resulting in the generation of polymers of adenosine diphosphate-ribose (PAR). Excessive linkage of PAR to nuclear proteins can induce cell death, thereby limiting the function of transplanted organs. This study uses a rat model of brain death to determine the profile of PARP activation and whether mechanisms that lead to cell death can be ameliorated by appropriate donor resuscitation. The expression of PAR-linked nuclear proteins within cardiac myocytes was greatly increased after the induction of donor brain death. Importantly, infusion of noradrenaline or vasopressin to normalize the chronic hypotension produced by brain death reduced the expression of PAR to a level below baseline. These data suggest that chronic hypotension after donor brain death has the potential to limit cardiac function through the activation of PARP; however, this early cause of graft damage can be mitigated by appropriate donor resuscitation.

  13. Xylazine Activates Adenosine Monophosphate-Activated Protein Kinase Pathway in the Central Nervous System of Rats

    Shi, Xing-Xing; Yin, Bai-Shuang; Yang, Peng; Chen, Hao; Li, Xin; Su, Li-Xue; Fan, Hong-Gang; Wang, Hong-Bin


    Xylazine is a potent analgesic extensively used in veterinary and animal experimentation. Evidence exists that the analgesic effect can be inhibited using adenosine 5’-monophosphate activated protein kinase (AMPK) inhibitors. Considering this idea, the aim of this study was to investigate whether the AMPK signaling pathway is involved in the central analgesic mechanism of xylazine in the rat. Xylazine was administrated via the intraperitoneal route. Sprague-Dawley rats were sacrificed and the cerebral cortex, cerebellum, hippocampus, thalamus and brainstem were collected for determination of liver kinase B1 (LKB1) and AMPKα mRNA expression using quantitative real-time polymerase chain reaction (qPCR), and phosphorylated LKB1 and AMPKα levels using western blot. The results of our study showed that compared with the control group, xylazine induced significant increases in AMPK activity in the cerebral cortex, hippocampus, thalamus and cerebellum after rats received xylazine (P < 0.01). Increased AMPK activities were accompanied with increased phosphorylation levels of LKB1 in corresponding regions of rats. The protein levels of phosphorylated LKB1 and AMPKα in these regions returned or tended to return to control group levels. However, in the brainstem, phosphorylated LKB1 and AMPKα protein levels were decreased by xylazine compared with the control (P < 0.05). In conclusion, our data indicates that xylazine alters the activities of LKB1 and AMPK in the central nervous system of rats, which suggests that xylazine affects the regulatory signaling pathway of the analgesic mechanism in the rat brain. PMID:27049320

  14. Effect of adenosine on the supramolecular architecture and activity of 5-fluorouracil

    Singh, Udai P.; Kashyap, Sujata; Singh, Hari Ji; Mishra, Bhupesh Kumar; Roy, Partha; Chakraborty, Ajanta


    The reactions of adenosine (Ad) with 5-halouracils (5XU where X = F for 1, Cl for 2, Br for 3 and I for 4) resulted in the formation of co-crystals 1-4 in monoclinic with P21 space group. Despite of great variation in the halo substituent at the 5th position of the uracil, each structure contains the same number and same type of non-covalent interactions i.e., primary N-H⋯N, N-H⋯O, O-H⋯N, O-H⋯O hydrogen bonds and secondary C-H⋯O and X⋯O interactions within these motifs as well as with neighboring molecules. As compared to Ad the size of cavity increases in co-crystal 1 to accommodate the 5FU as a guest. With the variation of halogen from fluoro to iodo on the uracil, the orientation of the molecules remains the same with a slight difference in the dihedral angle in all the co-crystals 1-4. This study demonstrates that hydrogen-bonded interactions between adenosine and halouracils provide a supramolecular assembly to these co-crystals. Computational studies illustrate that the size of the halo substituents on uracil has no effect on the hydrogen bond interaction energy. It further reveals that the orientation of molecules remain same in both solid phase as well as in the gaseous phase. The antitumor and DNA cleavage activity studies show that the antitumor activity of 5-fluorouracil against MCF-7 breast cancer decreases in the presence of adenosine.

  15. The effects of caffeine on sleep in Drosophila require PKA activity, but not the adenosine receptor.

    Wu, Mark N; Ho, Karen; Crocker, Amanda; Yue, Zhifeng; Koh, Kyunghee; Sehgal, Amita


    Caffeine is one of the most widely consumed stimulants in the world and has been proposed to promote wakefulness by antagonizing function of the adenosine A2A receptor. Here, we show that chronic administration of caffeine reduces and fragments sleep in Drosophila and also lengthens circadian period. To identify the mechanisms underlying these effects of caffeine, we first generated mutants of the only known adenosine receptor in flies (dAdoR), which by sequence is most similar to the mammalian A2A receptor. Mutants lacking dAdoR have normal amounts of baseline sleep, as well as normal homeostatic responses to sleep deprivation. Surprisingly, these mutants respond normally to caffeine. On the other hand, the effects of caffeine on sleep and circadian rhythms are mimicked by a potent phosphodiesterase inhibitor, IBMX (3-isobutyl-1-methylxanthine). Using in vivo fluorescence resonance energy transfer imaging, we find that caffeine induces widespread increase in cAMP levels throughout the brain. Finally, the effects of caffeine on sleep are blocked in flies that have reduced neuronal PKA activity. We suggest that chronic administration of caffeine promotes wakefulness in Drosophila, at least in part, by inhibiting cAMP phosphodiesterase activity.

  16. Modulatory effect of iron chelators on adenosine deaminase activity and gene expression in Trichomonas vaginalis.

    Primon-Barros, Muriel; Rigo, Graziela Vargas; Frasson, Amanda Piccoli; Santos, Odelta dos; Smiderle, Lisiane; Almeida, Silvana; Macedo, Alexandre José; Tasca, Tiana


    Trichomonas vaginalis is a flagellate protozoan that parasitises the urogenital human tract and causes trichomoniasis. During the infection, the acquisition of nutrients, such as iron and purine and pyrimidine nucleosides, is essential for the survival of the parasite. The enzymes for purinergic signalling, including adenosine deaminase (ADA), which degrades adenosine to inosine, have been characterised in T. vaginalis. In the evaluation of the ADA profile in different T. vaginalis isolates treated with different iron sources or with limited iron availability, a decrease in activity and an increase in ADA gene expression after iron limitation by 2,2-bipyridyl and ferrozine chelators were observed. This supported the hypothesis that iron can modulate the activity of the enzymes involved in purinergic signalling. Under bovine serum limitation conditions, no significant differences were observed. The results obtained in this study allow for the assessment of important aspects of ADA and contribute to a better understanding of the purinergic system in T. vaginalis and the role of iron in establishing infection and parasite survival.

  17. Amplified Peroxidase-Like Activity in Iron Oxide Nanoparticles Using Adenosine Monophosphate: Application to Urinary Protein Sensing.

    Yang, Ya-Chun; Wang, Yen-Ting; Tseng, Wei-Lung


    Numerous compounds such as protein and double-stranded DNA have been shown to efficiently inhibit intrinsic peroxidase-mimic activity in Fe3O4 nanoparticles (NP) and other related nanomaterials. However, only a few studies have focused on finding new compounds for enhancing the catalytic activity of Fe3O4 NP-related nanomaterials. Herein, phosphate containing adenosine analogs are reported to enhance the oxidation reaction of hydrogen peroxide (H2O2) and amplex ultrared (AU) for improving the peroxidase-like activity in Fe3O4 NPs. This enhancement is suggested to be a result of the binding of adenosine analogs to Fe(2+)/Fe(3+) sites on the NP surface and from adenosine 5'-monophosphate (AMP) acting as the distal histidine residue of horseradish peroxidase for activating H2O2. Phosphate containing adenosine analogs revealed the following trend for the enhanced activity of Fe3O4 NPs: AMP > adenosine 5'-diphosphate > adenosine 5'-triphosphate. The peroxidase-like activity in the Fe3O4 NPs progressively increased with increasing AMP concentration and polyadenosine length. The Michaelis constant for AMP attached Fe3O4 NPs is 5.3-fold lower and the maximum velocity is 2.7-fold higher than those of the bare Fe3O4 NPs. Furthermore, on the basis of AMP promoted peroxidase mimicking activity in the Fe3O4 NPs and the adsorption of protein on the NP surface, a selective fluorescent turn-off system for the detection of urinary protein is developed.

  18. Endogenous activation of adenosine A(1) receptors accelerates ischemic suppression of spontaneous electrocortical activity

    Ilie, Andrei; Ciocan, Dragos; Zagrean, Ana-Maria;


    Cerebral ischemia induces a rapid suppression of spontaneous brain rhythms prior to major alterations in ionic homeostasis. It was found in vitro during ischemia that the rapidly formed adenosine, resulting from the intracellular breakdown of ATP, may inhibit synaptic transmission via the A(1) re...

  19. Cerebrospinal fluid adenosine deaminase activity: A complimentary tool in the early diagnosis of tuberculous meningitis

    Taori Girdhar M


    Full Text Available Abstract Background Tuberculous meningitis (TBM is the commonest form of neurotuberculosis caused by Mycobacterium tuberculosis bacilli (MTB. The diagnosis of TBM is often difficult. A reliable, cost-effective and rapid diagnostic test, which can be performed in any standard pathology laboratory, could be of help in the diagnosis of TBM. In the present study we measured the adenosine deaminase (ADA activity in cerebrospinal fluid (CSF of TBM and non-TBM patients. Method ADA activity in CSF was determined according to a method based on the Berthlot reaction, which is the formation of a colored indophenol complex from ammonia liberated from adenosine, and quantified spectrophotometrically. Results The CSF ADA activity from TBM patients was compared with CSF ADA from non-TBM infectious meningitis patients, and from patients with non-infectious neurological disorders. The mean CSF ADA activity was found to be significantly higher in CSF of TBM patients, 14.31 ± 3.87 (2.99–26.94, mean ± SD with range, than in the CSF from non-TBM infectious meningitis, 9.25 ± 2.14 (4.99–13.96 and from the non-infectious neurological disorders group, 2.71 ± 1.96 (0.00–7.68, P Conclusion This study demonstrated that ADA activity in the CSF of TBM patients, using a cut-off value 11.39 U/L/min, can be useful for the early differential diagnosis of TBM. This test can be performed in any pathology laboratory where more sophisticated methods are not available.

  20. Serum Adenosine deaminase activity and C-reactive protein levels in unstable angina

    Rani Surekha


    Full Text Available In unstable angina (USA patients, immunological responses contributing to inflammation play a vital role in plaque rupture and thrombosis causing stroke. In the present study an attempt is made to estimate the levels of adenosine deaminase activity, an immunoenzyme marker and C-reactive protein, a marker of inflammation in USA patients. 45 patients presenting USA and 50 age and sex matched healthy controls were included in the study. Serum ADA activity was measured spectrophotometrically at 630nm and serum C-reactive protein was detected using Avitex CRP kit, which is a rapid latex agglutination test. The Mean ADA levels were 41.15 ± 11.04 in patients and 20.71±5.63 in controls and 66.6% of patients and none of the controls were positive to CRP. The present study observed the importance of ADA as a serum marker in addition to CRP for assessing the immune response in USA patients.

  1. Metabolic syndrome: adenosine monophosphate-activated protein kinase and malonyl coenzyme A.

    Ruderman, Neil B; Saha, Asish K


    The metabolic syndrome can be defined as a state of metabolic dysregulation characterized by insulin resistance, central obesity, and a predisposition to type 2 diabetes, dyslipidemia, premature atherosclerosis, and other diseases. An increasing body of evidence has linked the metabolic syndrome to abnormalities in lipid metabolism that ultimately lead to cellular dysfunction. We review here the hypothesis that, in many instances, the cause of these lipid abnormalities could be a dysregulation of the adenosine monophosphate-activated protein kinase (AMPK)/malonyl coenzyme A (CoA) fuel-sensing and signaling mechanism. Such dysregulation could be reflected by isolated increases in malonyl CoA or by concurrent changes in malonyl CoA and AMPK, both of which would alter intracellular fatty acid partitioning. The possibility is also raised that pharmacological agents and other factors that activate AMPK and/or decrease malonyl CoA could be therapeutic targets.

  2. Stabilizing effects of G protein on the active conformation of adenosine A1 receptor differ depending on G protein type.

    Tateyama, Michihiro; Kubo, Yoshihiro


    G protein coupled receptors (GPCRs) trigger various cellular and physiological responses upon the ligand binding. The ligand binding induces conformational change in GPCRs which allows G protein to interact with the receptor. The interaction of G protein also affects the active conformation of GPCRs. In this study, we have investigated the effects of Gαi1, Gαo and chimeric Gαqi5 on the active conformation of the adenosine A1 receptor, as each Gα showed difference in the interaction with adenosine A1 receptor. The conformational changes in the adenosine A1 receptor were detected as the agonist-induced decreases in efficiency of Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) fused at the two intracellular domains of the adenosine A1 receptor. Amplitudes of the agonist-induced FRET decreases were subtle when the FP-tagged adenosine A1 receptor was expressed alone, whereas they were significantly enhanced when co-expressed with Gαi1Gβ1Gγ22 (Gi1) or Gαqi5Gβ1Gγ22 (Gqi5) but not with GαοGβ1Gγ22 (Go). The enhancement of the agonist-induced FRET decrease in the presence of Gqi5 was significantly larger than that of Gi1. Furthermore, the FRET recovery upon the agonist removal in the presence of Gqi5 was significantly slower than that of Gi1. From these results it was revealed that the agonist-bound active conformation of adenosine A1 receptor is unstable without the binding of G protein and that the stabilizing effects of G protein differ depending on the types of G protein.

  3. The investigation of Adenosine Deaminase activity in patients with Mycosis Fungoides

    Yılmaz Ulaş


    Full Text Available Aim: Mycosis fungoides (MF is a cutaneous T cell lymphoma. The clinical and histophological diagnosis of early mycosis fungoides is usually diffucult. There is no special laboratory method for the diagnosis of MF disease and this is the most important problem in diagnosis and also follow up the effectiveness of treatment. Adenosine deaminase (ADA activity is a non-specific marker of T cell activation. In the present study, we aimed to investigate the levels of plasma and tissue ADA in patients with mycosis fungoides and to determine if ADA is an activation criteria for this disease. Materials and Medhods: The levels of ADA activities in both plasma and tissues were spectrophotometrically measured in 40 patients with MF and compared to those of 33 healthy subjects. Moreover, a subgroup analysis regarding ADA activities was performed in 17 patients who achieved complete remission after different kinds of treatments. Results: Patients with MF had more significantly elevated plasma and tissue ADA activity levels than those of control groups (respectively p0.05; MF patients in remission were found to have higher plasma levels of ADA activities than those of controls (p<0.001. Conclusion: These findings of the current study may provide an important clinical support for showing the roles of plasma and tissue ADA activity levels to predict disease activity in MF patients. In addition, levels of ADA activity measurements might be a marker to follow up in MF patients.

  4. Importance of mammalian nuclear-envelope nucleoside triphosphatase in nucleo-cytoplasmic transport of ribonucleoproteins.

    Agutter, P S; McCaldin, B; McArdle, H J


    The nucleoside triphosphate-stimulated efflux of RNA from isolated nuclei was studied under a range of conditions, and the effects of these conditions on the process were compared with the properties of the nucleoside triphosphatase located in the pore complex. A marked similarity between the rate of efflux and the rate of nucleoside triphosphate hydrolysis was apparent, in terms of substrate specificity, sensitivity to treatment with insolubilized trypsin, kinetics and the effects of increased ionic strength and of many inhibitors. These results are taken, in view of earlier evidence, to suggest that the activity of the nucleoside triphosphatase is a prerequisite for nucleo-cytoplasmic RNA transport in vivo. There are some indications that the nuclear-envelope lipid is also involved in regulating the efflux process.

  5. Does adenosine deaminase activity play a role in the early diagnosis of ectopic pregnancy?

    Turkmen, G G; Karçaaltıncaba, D; Isık, H; Fidancı, V; Kaayalp, D; Tımur, H; Batıoglu, S


    Early diagnosis of ectopic pregnancy (EP) is important due to life-threatening consequences in the first trimester of pregnancy. In this study we aimed to investigate the role of adenosine deaminase (ADA) activity in the prediction of EP. Forty-one patients with unruptured ectopic pregnancy comprised the case group and forty-two first trimester pregnant women with shown foetal heart beating in ultrasound comprised the control group. The mean ADA level in EP (10.9 ± 3.0 IU/L) was higher than that in control group (9.2 ± 3.6 IU/L) (p = 0.018). Receiver operating characteristics or ROC curve identified ADA value of 10.95 IU/L as optimal threshold for the prediction of EP with 56% sensitivity and 67% specificity. High ADA levels are valuable in the early diagnosis of EP. However more comprehensive studies are required.

  6. Elevated erythrocyte adenosine deaminase activity in a patient with primary acquired sideroblastic anemia.

    Kanno, H; Fujii, H; Tani, K; Morisaki, T; Takahashi, K; Horiuchi, N; Kizaki, M; Ogawa, T; Miwa, S


    We report a case of primary acquired sideroblastic anemia (PASA) associated with elevated erythrocyte adenosine deaminase (ADA) activity. The patient was an 85-year-old Japanese male. Analysis of the peripheral blood revealed pancytopenia, and the bone marrow findings showed marked ringed sideroblasts and chromosomal deletion (46XY, 11q-). The erythrocyte ADA activity was 17 times higher than that of normal control, the leukocyte ADA activity was within the normal range, and the plasma ADA activity was 2 times higher than the normal mean. The adenine nucleotides in the patient's erythrocytes were within normal range. According to starch gel electrophoresis, ADA isozyme of the patient was ADA 1. Western blotting showed an increased amount of ADA protein in the patient's erythrocytes. Southern blotting revealed no gene amplification or large structural change. Dot blot analysis of the reticulocyte mRNA showed no increase in the amount of ADA mRNA in the patient's reticulocytes compared with those of reticulocyte-rich controls. We considered that the mechanism of elevated ADA activity in this acquired defect was similar to that found in hereditary hemolytic anemia associated with ADA overproduction.

  7. Synthesis and pharmacological characterization of novel xanthine carboxylate amides as A2A adenosine receptor ligands exhibiting bronchospasmolytic activity.

    Yadav, Rakesh; Bansal, Ranju; Rohilla, Suman; Kachler, Sonja; Klotz, Karl-Norbert


    The carboxylate amides of 8-phenyl-1,3-dimethylxanthine described herein represent a new series of selective ligands of the adenosine A2A receptors exhibiting bronchospasmolytic activity. The effects of location of 8-phenyl substitutions on the adenosine receptor (AR) binding affinities of the newly synthesized xanthines have also been studied. The compounds displayed moderate to potent binding affinities toward various adenosine receptor subtypes when evaluated through radioligand binding studies. However, most of the compounds showed the maximum affinity for the A2A subtype, some with high selectivity versus all other subtypes. Xanthine carboxylate amide 13b with a diethylaminoethylamino moiety at the para-position of the 8-phenylxanthine scaffold was identified as the most potent A2A adenosine receptor ligand with Ki=0.06μM. Similarly potent and highly A2A-selective are the isovanillin derivatives 16a and 16d. In addition, the newly synthesized xanthine derivatives showed good in vivo bronchospasmolytic activity when tested in guinea pigs.

  8. 5'-adenosine monophosphate-activated protein kinase and the metabolic syndrome.

    Mor, Vijay; Unnikrishnan, M K


    Lifestyle changes such as physical inactivity combined with calorie-rich, low-fibre diets have triggered an explosive surge in metabolic syndrome, outlined as a cluster of heart attack risk factors such as insulin resistance, raised fasting plasma glucose, abdominal obesity, high cholesterol and high blood pressure. By acting as a master-switch of energy homeostasis and associated pathophysiological phenomena, 5'-adenosine monophosphate-activated protein kinase (AMPK) appears to orchestrate the adaptive physiology of energy deficit, suggesting that the sedentary modern human could be suffering from chronic suboptimal AMPK activation. Addressing individual targets with potent ligands with high specificity may be inappropriate (it has not yielded any molecule superior to the sixty year old metformin) because this strategy cannot address a cluster of interrelated pathologies. However, spices, dietary supplements and nutraceuticals attenuate the multiple symptoms of metabolic syndrome in a collective and perhaps more holistic fashion with fewer adverse events. Natural selection could have favoured races that developed a taste for spices and dietary supplements, most of which are not only antioxidants but also activators of AMPK. The review will outline the various biochemical mechanisms and pathophysiological consequences of AMPK activation involving the cluster of symptoms that embrace metabolic syndrome and beyond. Recent advances that integrate energy homeostasis with a number of overarching metabolic pathways and physiological phenomena, including inflammatory conditions, cell growth and development, malignancy, life span, and even extending into environmental millieu, as in obesity mediated by gut microflora and others will also be outlined.

  9. Effects of adenosine 5’monophosphate-activated protein kinase on europrotection induced by ischemic preconditioning

    Yuan-ru-hua TIAN


    Full Text Available Objective To investigate the effects of adenosine 5'-monophosphate-activated protein kinase (AMPK and phosphated AMPK (pAMPK signals in ischemic preconditioning (IPC, and the effect of pharmacological intervention of AMPK on infarct size of the brain. Methods A brief (3min middle cerebral artery occlusion (MCAO was employed to induce IPC in male rat, and another 90-min MCAO was performed 4 or 72h later. The levels of AMPK and pAMPK were assessed after IPC. A pharmacological activator metformin, or inhibitor compound C of AMPK, was used to analyze the correlation of IPC to AMPK signaling in MCAO rats. Results The infarct size of total cerebral hemisphere and cortex was significantly decreased in MCAO animals by IPC for 72h (P0.05, n=6. The AMPK activator metformin can significantly reverse the protective effect of IPC (P<0.05, n=6. Conclusions The signals of AMPK and pAMPK play an important role in neuroprotective effect of IPC on cerebral ischemic injury. The neuroprotective effect of IPC may be associated with the down-regulation of pAMPK. DOI: 10.11855/j.issn.0577-7402.2015.05.07

  10. Adenosine deaminase activity in serum, erythrocytes and lymphocytes of rats infected with Leptospira icterohaemorrhagiae.

    Tonin, Alexandre A; Pimentel, Victor C; da Silva, Aleksandro S; de Azevedo, Maria Isabel; Souza, Viviane C G; Wolkmer, Patrícia; Rezer, João F P; Badke, Manoel R T; Leal, Daniela B R; Schetinger, Maria Rosa C; Monteiro, Silvia G; Lopes, Sonia T A


    Leptospirosis is a systemic disease of humans and domestic animals, mainly dogs, cattle and swine. The course of human leptospirosis varies from mild to severe fatal forms and the most severe form of human leptospirosis is principally caused by Leptospira interrogans serovar icterohaemorrhagiae (L. icterohaemorrhagiae). The enzyme adenosine deaminase (ADA) plays an important role in the production and differentiation of blood cells. The aim of this study was to evaluate the activity of ADA in serum, erythrocytes and lymphocytes of rats infected with L. icterohaemorrhagiae, as compared with non-infected rats. Twenty-four adult rats, divided into two uniform groups (A and B) were used for the enzymatic assays. The animals in Group B were inoculated intraperitoneally with 2×10(8) leptospires/rat, and the rodents in Group A (control) were not-inoculated. Blood collection was performed on days 5 and 15 post-infection (PI) and the blood used to assess the ADA activity. The infection by L.icterohaemorrhagiae altered erythrocyte count, hemoglobin concentration and hematocrit, causing a decrease in all these parameters on day 15 PI. Lymphocytes decreased significantly on day 15 PI, and ADA activity in serum was inhibited in infected rats on days 5 and 15 PI and its activity in erythrocytes were increased on day 5 PI. On day 5 PI, we found an increase in ADA activity in erythrocytes of infected rats. No correlation was observed between hematocrit and erythrocyte ADA activity on days 5 and 15 PI. The ADA activity was inhibited in rats infected on day 15 PI. A positive correlation (r(2)=60) was also observed between the number of lymphocytes and ADA activity in lymphocytes on day 15 PI (Perythrocytes in experimental infection by L.icterohaemorrhagiae in rats, concomitantly with hematological parameters.

  11. Anticonvulsant effect of AMP by direct activation of adenosine A1 receptor.

    Muzzi, Mirko; Coppi, Elisabetta; Pugliese, Anna Maria; Chiarugi, Alberto


    Purinergic neurotransmission mediated by adenosine (Ado) type 1 receptors (A1Rs) plays pivotal roles in negative modulation of epileptic seizures, and Ado is thought to be a key endogenous anticonvulsant. Recent evidence, however, indicates that AMP, the metabolic precursor of Ado, also activate A1Rs. Here, we evaluated the antiepileptic effects of AMP adopting in vitro and in vivo models of epilepsy. We report that AMP reversed the increase in population spike (PS) amplitude and the decrease in PS latency induced by a Mg(2+)-free extracellular solution in CA1 neurons of mouse hippocampal slices. The AMP effects were inhibited by the A1R antagonist DPCPX, but not prevented by inhibiting conversion of AMP into Ado, indicating that AMP inhibited per se sustained hippocampal excitatory neurotransmission by directly activating A1Rs. AMP also reduced seizure severity and mortality in a model of audiogenic convulsion. Of note, the anticonvulsant effects of AMP were potentiated by preventing its conversion into Ado and inhibited by DPCPX. When tested in a model of kainate-induced seizure, AMP prolonged latency of convulsions but had no effects on seizure severity and mortality. Data provide the first evidence that AMP is an endogenous anticonvulsant acting at A1Rs.

  12. Fine mapping and functional activity of the adenosine deaminase origin in murine embryonic fibroblasts.

    Sibani, Sahar; Rampakakis, Emmanouil; Di Paola, Domenic; Zannis-Hadjopoulos, Maria


    DNA replication initiates at origins within the genome. The late-firing murine adenosine deaminase (mAdA) origin is located within a 2 kb fragment of DNA, making it difficult to examine by realtime technology. In this study, fine mapping of the mAdA region by measuring the abundance of nascent strand DNA identified two origins, mAdA-1 and mAdA-C, located 397 bp apart from each other. Both origins conferred autonomous replication to plasmids transfected in murine embryonic fibroblasts (MEFs), and exhibited similar activities in vivo and in vitro. Furthermore, both were able to recruit the DNA replication initiator proteins Cdc6 and Ku in vitro, similar to other bona fide replication origins. When tested in a murine Ku80(-/-) cell line, both origins exhibited replication activities comparable to those observed in wildtype cells, as did the hypoxanthine-guanine phosphoribosyltransferase (HPRT) and c-myc origins. This contrasts with previously published studies using Ku80-deficient human cells lines and suggests differences in the mechanism of initiation of DNA replication between the murine and human systems.

  13. Promotion of Wound Healing by an Agonist of Adenosine A2A Receptor Is Dependent on Tissue Plasminogen Activator.

    Montesinos, M Carmen; Desai-Merchant, Avani; Cronstein, Bruce N


    Impaired wound healing, as it occurs in diabetes mellitus or long-term corticoid treatment, is commonly associated with disability, diminished quality of life, and high economic costs. Selective agonists of the A2A receptor subtype of adenosine, an endogenous regulator of inflammation, promote tissue repair in animal models, both healthy and with impaired healing. Plasmin-mediated proteolysis of fibrin and other matrix proteins is essential for cell migration at sites of injury. Since adenosine A2A receptor activation increases plasminogen activator release from macrophages and mast cells, we studied the effect of a selective agonist, CGS-21680, on full-thickness excisional wound closure in wild-type, urokinase plasminogen activator (uPA)-deficient, and tissue plasminogen activator (tPA)-deficient mice. Wound closure was impaired in tPA- and uPA-deficient mice as compared with wild-type mice, and topical application of CGS-21680 significantly increased the rate at which wounds closed in wild-type mice and uPA-deficient mice, but not in tPA-deficient mice. Immunostaining of tissue sections showed that tPA was present in endothelial cells and histiocytes by day 3 post-wound and also by day 6. In contrast, uPA was more prominent in these cell types only by day 6 post-wound. Our results confirm that plasminogen activation contributes to wound repair and are consistent with the hypothesis that adenosine A2A receptor activation promotes wound closure by a mechanism that depends upon tPA, but not uPA. Moreover, our results suggest that topical adenosine A2A receptor agonists may be useful in promotion of wound closure in patients with impaired wound healing.

  14. SNC-80-induced preconditioning: selective activation of the mitochondrial adenosine triphosphate-gated potassium channel.

    Fischbach, Peter S; Barrett, Terrance D; Reed, Nathan J; Lucchesi, Benedict R


    Pharmacologic preconditioning by delta-opioid agonists occurs via activation of an adenosine triphosphate (ATP)-gated potassium channel (I(KATP)). Opening of mitochondrial I(KATP) confers pharmacologic preconditioning whereas opening the sarcolemmal I(KATP) shortens action potential duration and is proarrhythmic. This study investigated whether SNC-80, a selective delta-opioid agonist, is associated with development of ventricular arrhythmia due to activation of I(KATP). Rabbit isolated hearts were subjected to 12 min of hypoxia and 40 min of reoxygenation after pretreatment with SNC-80 (1 microM, n = 6), pinacidil (1.25 microM, n = 12), or BMS-191095 (6.0 microM, n = 4). Nine additional hearts served as controls. The cytoprotective effects of SNC-80 at a concentration of 1 microM were confirmed using 30 min of regional ischemia followed by 120 min of reperfusion. Ventricular fibrillation (VF) developed in 11 of 12 pinacidil-treated hearts whereas none of the SNC-80-treated (zero of six) hearts developed VF (P SNC-80 reduced infarct size expressed as a percentage of the area at risk from 33 +/- 4% to 14 +/- 3% (P = 0.004) compared with control. SNC-80, which selectively activates the delta-opioid receptor, provided cytoprotection but did not induce VF after hypoxia reoxygenation. The results indicate that pinacidil-induced nonselective activation of I(KATP) results in proarrhythmia that is dependent on activation of the sarcolemmal I(KATP). Selectivity for the mitochondrial I(KATP) is necessary to prevent induction of a proarrhythmic state.

  15. Increased orbitofrontal brain activation after administration of a selective adenosine A2A antagonist in cocaine dependent subjects

    F. Gerard eMoeller


    Full Text Available Background: Positron Emission Tomography imaging studies provide evidence of reduced dopamine function in cocaine dependent subjects in the striatum, which is correlated with prefrontal cortical glucose metabolism, particularly in the orbitofrontal cortex. However, whether enhancement of dopamine in the striatum in cocaine dependent subjects would be associated with changes in prefrontal cortical brain activation is unknown. One novel class of medications that enhance dopamine function via heteromer formation with dopamine receptors in the striatum is the selective adenosine A2A receptor antagonists. This study sought to determine the effects administration of the selective adenosine A2A receptor antagonist SYN115 on brain function in cocaine dependent subjects. Methodology/Principle Findings: Twelve cocaine dependent subjects underwent two fMRI scans (one after a dose of placebo and one after a dose of 100 mg of SYN115 while performing a working memory task with 3 levels of difficulty (3, 5, and 7 digits. fMRI results showed that for 7-digit working memory activation there was significantly greater activation from SYN115 compared to placebo in portions of left (L lateral orbitofrontal cortex, L insula, and L superior and middle temporal pole. Conclusion/Significance: These findings are consistent with enhanced dopamine function in the striatum in cocaine dependent subjects via blockade of adenosine A2A receptors producing increased brain activation in the orbitofrontal cortex and other cortical regions. This suggests that at least some of the changes in brain activation in prefrontal cortical regions in cocaine dependent subjects may be related to altered striatal dopamine function, and that enhancement of dopamine function via adenosine A2A receptor blockade could be explored further for amelioration of neurobehavioral deficits associated with chronic cocaine use.


    Iván Darío BRAVO-TOBAR


    Full Text Available SUMMARY Chagas disease is a public health problem worldwide. The availability of diagnostic tools to predict the development of chronic Chagas cardiomyopathy is crucial to reduce morbidity and mortality. Here we analyze the prognostic value of adenosine deaminase serum activity (ADA and C-reactive protein serum levels (CRP in chagasic individuals. One hundred and ten individuals, 28 healthy and 82 chagasic patients were divided according to disease severity in phase I (n = 35, II (n = 29, and III (n = 18. A complete medical history, 12-lead electrocardiogram, chest X-ray, and M-mode echocardiogram were performed on each individual. Diagnosis of Chagas disease was confirmed by ELISA and MABA using recombinant antigens; ADA was determined spectrophotometrically and CRP by ELISA. The results have shown that CRP and ADA increased linearly in relation to disease phase, CRP being significantly higher in phase III and ADA at all phases. Also, CRP and ADA were positively correlated with echocardiographic parameters of cardiac remodeling and with electrocardiographic abnormalities, and negatively with ejection fraction. CRP and ADA were higher in patients with cardiothoracic index ≥ 50%, while ADA was higher in patients with ventricular repolarization disturbances. Finally, CRP was positively correlated with ADA. In conclusion, ADA and CRP are prognostic markers of cardiac dysfunction and remodeling in Chagas disease.

  17. Diagnostic value of pleural fluid adenosine deaminase activity in tuberculosis pleurisy

    Abbas ali Niazi


    Full Text Available Background: Diagnosis of tuberculosis pleurisies is difficult because of its nonspecific clinical presentation and insufficient traditional diagnostic methods. We investigated the use of adenosine deaminase (ADA activity in tuberculosis pleurisies. Methods: A number of 85 patients were analyzed with exudative pleural effusions. Using the ROC curve, we determined the optimal cutoff for TB pleurisy. Results: A number of 58 exudative samples were nontuberculous (non-TB and 27 were tuberculosis (TB. There was statistically significant difference (p<0.0001 between the means of pleural fluid ADA levels among the TB and non-TB populations. The prevalence of TB pleurisy in the studied population was 31%. Using the cutoff point equal to 35 for diagnosing TB effusions the sensitivity and specificity 70.3% and 91.3%, respectively. The positive predictive value (PPV was 79.1% and the negative predictive value (NPV was 86.8%. A pleural fluid ADA value <19 IU/L suggests that a tuberculosis effusion is highly unlikely. Conclusion: Pleural fluid total ADA assay is a sensitive and specific test suitable for rapid diagnosis of TB pleurisy.




    SUMMARY Chagas disease is a public health problem worldwide. The availability of diagnostic tools to predict the development of chronic Chagas cardiomyopathy is crucial to reduce morbidity and mortality. Here we analyze the prognostic value of adenosine deaminase serum activity (ADA) and C-reactive protein serum levels (CRP) in chagasic individuals. One hundred and ten individuals, 28 healthy and 82 chagasic patients were divided according to disease severity in phase I (n = 35), II (n = 29), and III (n = 18). A complete medical history, 12-lead electrocardiogram, chest X-ray, and M-mode echocardiogram were performed on each individual. Diagnosis of Chagas disease was confirmed by ELISA and MABA using recombinant antigens; ADA was determined spectrophotometrically and CRP by ELISA. The results have shown that CRP and ADA increased linearly in relation to disease phase, CRP being significantly higher in phase III and ADA at all phases. Also, CRP and ADA were positively correlated with echocardiographic parameters of cardiac remodeling and with electrocardiographic abnormalities, and negatively with ejection fraction. CRP and ADA were higher in patients with cardiothoracic index ≥ 50%, while ADA was higher in patients with ventricular repolarization disturbances. Finally, CRP was positively correlated with ADA. In conclusion, ADA and CRP are prognostic markers of cardiac dysfunction and remodeling in Chagas disease. PMID:26603224

  19. Adenosine activates ATP-sensitive potassium channels in arterial myocytes via A2 receptors and cAMP-dependent protein kinase.

    Kleppisch, T; Nelson, M T


    The mechanism by which the endogenous vasodilator adenosine causes ATP-sensitive potassium (KATP) channels in arterial smooth muscle to open was investigated by the whole-cell patch-clamp technique. Adenosine induced voltage-independent, potassium-selective currents, which were inhibited by glibenclamide, a blocker of KATP currents. Glibenclamide-sensitive currents were also activated by the selective adenosine A2-receptor agonist 2-p-(2-carboxethyl)-phenethylamino-5'-N- ethylcarboxamidoadenosine hydrochloride (CGS-21680), whereas 2-chloro-N6-cyclopentyladenosine (CCPA), a selective adenosine A1-receptor agonist, failed to induce potassium currents. Glibenclamide-sensitive currents induced by adenosine and CGS-21680 were largely reduced by blockers of the cAMP-dependent protein kinase (Rp-cAMP[S], H-89, protein kinase A inhibitor peptide). Therefore, we conclude that adenosine can activate KATP currents in arterial smooth muscle through the following pathway: (i) Adenosine stimulates A2 receptors, which activates adenylyl cyclase; (ii) the resulting increase intracellular cAMP stimulates protein kinase A, which, probably through a phosphorylation step, opens KATP channels. PMID:8618917

  20. Adenosine monophosphate-activated protein kinase activation and suppression of inflammatory response by cell stretching in rabbit synovial fibroblasts.

    Kunanusornchai, Wanlop; Muanprasat, Chatchai; Chatsudthipong, Varanuj


    Joint mobilization is known to be beneficial in osteoarthritis (OA) patients. This study aimed to investigate the effect of stretching on adenosine monophosphate-activated protein kinase (AMPK) activity and its role in modulating inflammation in rabbit synovial fibroblasts. Uniaxial stretching of isolated rabbit synovial fibroblasts for ten min was performed. Stretching-induced AMPK activation, its underlying mechanism, and its anti-inflammatory effect were investigated using Western blot. Static stretching at 20 % of initial length resulted in AMPK activation characterized by expression of phosphorylated AMPK and phosphorylated acetyl-Co A carboxylase. AMP-activated protein kinase phosphorylation peaked 1 h after stretching and declined toward resting activity. Using cell viability assays, static stretching did not appear to cause cellular damage. Activation of AMPK involves Ca(2+) influx via a mechanosensitive L-type Ca(2+) channel, which subsequently raises intracellular Ca(2+) and activates AMPK via Ca(2+)/calmodulin-dependent protein kinase kinase β (CaMKKβ). Interestingly, stretching suppressed TNFα-induced expression of COX-2, iNOS, and phosphorylated NF-κB. These effects were prevented by pretreatment with compound C, an AMPK inhibitor. These results suggest that mechanical stretching suppressed inflammatory responses in synovial fibroblasts via a L-type Ca(2+)-channel-CaMKKβ-AMPK-dependent pathway which may underlie joint mobilization's ability to alleviate OA symptoms.

  1. Evaluation of adenosine deaminase seric activity in the diagnosis of bovine tuberculosis

    Márcio Roberto Silva


    Full Text Available Determination of seric levels of adenosine deaminase (ADA, an enzyme produced by monocytes/macrophages and lymphocytes, has been used in the diagnosis of human tuberculosis (TB. In the present study, ADA seric activity was evaluated comparatively to the comparative tuberculin test in the diagnosis of bovine tuberculosis. Two hundred fifty-six cattle were classified by origin and by the comparative tuberculin test as TB-positive animals (n = 52, from herds where the Mycobacterium bovis had previously been isolated, and TB-negative animals (n = 204, TB-free herds. The mean ADA seric value from the TB-positive group (4.45 ± 2.33 U/L was significantly lower (p = 0.008 than that observed in sera from the TB-negative group (6.12 ± 4.47 U/L. When animals from a herd with clinical cases of enzootic bovine leukosis of TB-negative group were withdrawn from analysis, the mean ADA seric values of TB-negative group (5.12 ± 3.75 U/L was not significantly different anymore from that of the TB-positive group (p = 0.28. There was no agreement in the diagnosis of bovine TB between comparative tuberculin test and determination of ADA seric values, using two different cutoff points, being 6.12 U/L and 15.0 U/L, (kappa = -0.086 and kappa = -0.082, respectively. In conclusion, the determination of ADA seric activity was not a good auxiliary test for bovine TB, because it was not able to distinguish between TB-positive and TB-negative animals.

  2. [Substrate specificity and kinetic properties of a soluble nucleoside triphosphatase from bovine kidneys].

    Sivuk, V F; Rusina, I M; Luchko, T A; Makarchikov, A F


    Soluble nucleoside triphosphatase differing in its properties from all known proteins with NTPase activity was partially purified from bovine kidneys. The enzyme has pH optimum of 7.5, molecular mass of 60 kDa, as estimated by gel chromatography, and shows an absolute dependence on divalent metal ions. NTPase obeyed Michaelis-Menten kinetics in the range of substrate concentration tested from 45 to 440 microM; the apparent Km for inosine-5'-triphosphate was calculated to be 23.3 microM. The enzyme was found to possess a broad substrate specificity, being capable of hydrolyzing various nucleoside-5'-tri- as well as diphosphates.

  3. Creatine, similarly to ketamine, affords antidepressant-like effects in the tail suspension test via adenosine A₁ and A2A receptor activation.

    Cunha, Mauricio P; Pazini, Francis L; Rosa, Julia M; Ramos-Hryb, Ana B; Oliveira, Ágatha; Kaster, Manuella P; Rodrigues, Ana Lúcia S


    The benefits of creatine supplementation have been reported in a broad range of central nervous systems diseases, including depression. A previous study from our group demonstrated that creatine produces an antidepressant-like effect in the tail suspension test (TST), a predictive model of antidepressant activity. Since depression is associated with a dysfunction of the adenosinergic system, we investigated the involvement of adenosine A1 and A2A receptors in the antidepressant-like effect of creatine in the TST. The anti-immobility effect of creatine (1 mg/kg, po) or ketamine (a fast-acting antidepressant, 1 mg/kg, ip) in the TST was prevented by pretreatment of mice with caffeine (3 mg/kg, ip, nonselective adenosine receptor antagonist), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (2 mg/kg, ip, selective adenosine A1 receptor antagonist), and 4-(2-[7-amino-2-{2-furyl}{1,2,4}triazolo-{2,3-a}{1,3,5}triazin-5-yl-amino]ethyl)-phenol (ZM241385) (1 mg/kg, ip, selective adenosine A2A receptor antagonist). In addition, the combined administration of subeffective doses of creatine and adenosine (0.1 mg/kg, ip, nonselective adenosine receptor agonist) or inosine (0.1 mg/kg, ip, nucleoside formed by the breakdown of adenosine) reduced immobility time in the TST. Moreover, the administration of subeffective doses of creatine or ketamine combined with N-6-cyclohexyladenosine (CHA) (0.05 mg/kg, ip, selective adenosine A1 receptor agonist), N-6-[2-(3,5-dimethoxyphenyl)-2-(methylphenyl)ethyl]adenosine (DPMA) (0.1 mg/kg, ip, selective adenosine A2A receptor agonist), or dipyridamole (0.1 μg/mouse, icv, adenosine transporter inhibitor) produced a synergistic antidepressant-like effect in the TST. These results indicate that creatine, similarly to ketamine, exhibits antidepressant-like effect in the TST probably mediated by the activation of both adenosine A1 and A2A receptors, further reinforcing the potential of targeting the purinergic system to the management of mood disorders.

  4. Piracetam prevents scopolamine-induced memory impairment and decrease of NTPDase, 5'-nucleotidase and adenosine deaminase activities.

    Marisco, Patricia C; Carvalho, Fabiano B; Rosa, Michelle M; Girardi, Bruna A; Gutierres, Jessié M; Jaques, Jeandre A S; Salla, Ana P S; Pimentel, Víctor C; Schetinger, Maria Rosa C; Leal, Daniela B R; Mello, Carlos F; Rubin, Maribel A


    Piracetam improves cognitive function in animals and in human beings, but its mechanism of action is still not completely known. In the present study, we investigated whether enzymes involved in extracellular adenine nucleotide metabolism, adenosine triphosphate diphosphohydrolase (NTPDase), 5'-nucleotidase and adenosine deaminase (ADA) are affected by piracetam in the hippocampus and cerebral cortex of animals subjected to scopolamine-induced memory impairment. Piracetam (0.02 μmol/5 μL, intracerebroventricular, 60 min pre-training) prevented memory impairment induced by scopolamine (1 mg/kg, intraperitoneal, immediately post-training) in the inhibitory avoidance learning and in the object recognition task. Scopolamine reduced the activity of NTPDase in hippocampus (53 % for ATP and 53 % for ADP hydrolysis) and cerebral cortex (28 % for ATP hydrolysis). Scopolamine also decreased the activity of 5'-nucleotidase (43 %) and ADA (91 %) in hippocampus. The same effect was observed in the cerebral cortex for 5'-nucleotidase (38 %) and ADA (68 %) activities. Piracetam fully prevented scopolamine-induced memory impairment and decrease of NTPDase, 5'-nucleotidase and adenosine deaminase activities in synaptosomes from cerebral cortex and hippocampus. In vitro experiments show that piracetam and scopolamine did not alter enzymatic activity in cerebral cortex synaptosomes. Moreover, piracetam prevented scopolamine-induced increase of TBARS levels in hippocampus and cerebral cortex. These results suggest that piracetam-induced improvement of memory is associated with protection against oxidative stress and maintenance of NTPDase, 5'-nucleotidase and ADA activities, and suggest the purinergic system as a putative target of piracetam.

  5. Adenosine receptor neurobiology: overview.

    Chen, Jiang-Fan; Lee, Chien-fei; Chern, Yijuang


    Adenosine is a naturally occurring nucleoside that is distributed ubiquitously throughout the body as a metabolic intermediary. In the brain, adenosine functions as an important upstream neuromodulator of a broad spectrum of neurotransmitters, receptors, and signaling pathways. By acting through four G-protein-coupled receptors, adenosine contributes critically to homeostasis and neuromodulatory control of a variety of normal and abnormal brain functions, ranging from synaptic plasticity, to cognition, to sleep, to motor activity to neuroinflammation, and cell death. This review begun with an overview of the gene and genome structure and the expression pattern of adenosine receptors (ARs). We feature several new developments over the past decade in our understanding of AR functions in the brain, with special focus on the identification and characterization of canonical and noncanonical signaling pathways of ARs. We provide an update on functional insights from complementary genetic-knockout and pharmacological studies on the AR control of various brain functions. We also highlight several novel and recent developments of AR neurobiology, including (i) recent breakthrough in high resolution of three-dimension structure of adenosine A2A receptors (A2ARs) in several functional status, (ii) receptor-receptor heterodimerization, (iii) AR function in glial cells, and (iv) the druggability of AR. We concluded the review with the contention that these new developments extend and strengthen the support for A1 and A2ARs in brain as therapeutic targets for neurologic and psychiatric diseases.

  6. Presynaptic facilitatory adenosine A2A receptors mediate fade induced by neuromuscular relaxants that exhibit anticholinesterase activity.

    Bornia, Elaine Cs; Correia-de-Sá, Paulo; Alves-Do-Prado, Wilson


    1. Pancuronium, cisatracurium and vecuronium are antinicotinic agents that, in contrast with d-tubocurarine and hexamethonium, exhibit anticholinesterase activity. Pancuronium-, cisatracurium- and vecuronium-induced fade results from blockade of facilitatory nicotinic receptors on motor nerves, but fade produced by such agents also depends on the presynaptic activation of inhibitory muscarinic M2 receptors by acetylcholine released from motor nerve terminals and activation of inhibitory adenosine A1 receptors by adenosine released from motor nerves and muscles. The participation of presynaptic facilitatory A2A receptors in fade caused by pancuronium, cisatracurium and vecuronium has not yet been investigated. In the present study, we determined the effects of ZM241385, an antagonist of presynaptic facilitatory A2A receptors, on fade produced by these neuromuscular relaxants in the rat phrenic nerve-diaphragm (PND) preparation. 2. The muscles were stimulated indirectly at 75±3Hz to induce a sustained tetanizing muscular contraction. The lowest concentration at which each antinicotinic agent produced fade without modifying initial tetanic tension (presynaptic action) was determined. 3. d-Tubocurarine-induced fade occurred only at 55 nmol/L, a concentration that also reduced maximal tetanic tension (post-synaptic action). At 10 nmol/L, ZM 241385 alone did not produce fade, but it did attenuate pancuronium (0.32 μmol/L)-, cisatracurium (0.32 μmol/L)- and vecuronium (0.36 μmol/L)-induced fade. 4. The fade induced by the 'pure' antinicotinic agents d-tubocurarine (55 nmol/L) and hexamethonium (413 μmol/L) was not altered by 10 nmol/L ZM 241385, indicating that presynaptic adenosine A2A receptors play a significant role in the fade produced by antinicotinic agents when such agents have anticholinesterase activity.

  7. Effects of aqueous extract from Silybum marianum on adenosine deaminase activity in cancerous and noncancerous human gastric and colon tissues

    Bahadır Öztürk


    Full Text Available Objective: Investigation of possible effects of Silybum marianum extract (SME on adenosine deaminase (ADA activity in cancerous and noncancerous human gastric and colon tissues to obtain information about possible mechanism of anticancer action of S. marianum. Materials and Methods: Cancerous and noncancerous human gastric and colon tissues removed from patients by surgical operations were used in the studies. The extract was prepared in distilled water. Before and after treatment with the extract, ADA activities in the samples were measured. Results: ADA activity was found to be lowered significantly in cancerous gastric tissues but not in noncancerous gastric tissues after treatment with the SME. In the colon tissues, ADA activities were however found to increase after the treatment of SME. Conclusion: Our results suggest that the aqueous extract from S. marianum inhibits ADA activity in cancerous gastric tissues significantly. It is suggested that in addition to other proposed mechanisms, accumulated adenosine due to the inhibition of ADA might also play a part in the anticancer properties of the S. marianum.

  8. Adenosine can thwart antitumor immune responses elicited by radiotherapy. Therapeutic strategies alleviating protumor ADO activities

    Vaupel, Peter [Klinikum rechts der Isar, Technische Universitaet Muenchen (TUM), Department of Radiation Oncology, Munich (Germany); Multhoff, Gabriele [Klinikum rechts der Isar, Technische Universitaet Muenchen (TUM), Department of Radiation Oncology, Munich (Germany); Helmholtz Zentrum Muenchen, Institute for innovative Radiotherapy (iRT), Experimental Immune Biology, Neuherberg (Germany)


    By studying the bioenergetic status we could show that the development of tumor hypoxia is accompanied, apart from myriad other biologically relevant effects, by a substantial accumulation of adenosine (ADO). ADO has been shown to act as a strong immunosuppressive agent in tumors by modulating the innate and adaptive immune system. In contrast to ADO, standard radiotherapy (RT) can either stimulate or abrogate antitumor immune responses. Herein, we present ADO-mediated mechanisms that may thwart antitumor immune responses elicited by RT. An overview of the generation, accumulation, and ADO-related multifaceted inhibition of immune functions, contrasted with the antitumor immune effects of RT, is provided. Upon hypoxic stress, cancer cells release ATP into the extracellular space where nucleotides are converted into ADO by hypoxia-sensitive, membrane-bound ectoenzymes (CD39/CD73). ADO actions are mediated upon binding to surface receptors, mainly A2A receptors on tumor and immune cells. Receptor activation leads to a broad spectrum of strong immunosuppressive properties facilitating tumor escape from immune control. Mechanisms include (1) impaired activity of CD4 + T and CD8 + T, NK cells and dendritic cells (DC), decreased production of immuno-stimulatory lymphokines, and (2) activation of Treg cells, expansion of MDSCs, promotion of M2 macrophages, and increased activity of major immunosuppressive cytokines. In addition, ADO can directly stimulate tumor proliferation and angiogenesis. ADO mechanisms described can thwart antitumor immune responses elicited by RT. Therapeutic strategies alleviating tumor-promoting activities of ADO include respiratory hyperoxia or mild hyperthermia, inhibition of CD39/CD73 ectoenzymes or blockade of A2A receptors, and inhibition of ATP-release channels or ADO transporters. (orig.) [German] Untersuchungen des bioenergetischen Status ergaben, dass Tumorhypoxie neben vielen anderen bedeutsamen biologischen Effekten zu einem starken

  9. Trypanocidal activity of 8-methyl-5'-{[(Z)-4-aminobut-2-enyl]-(methylamino)}adenosine (Genz-644131), an adenosylmethionine decarboxylase inhibitor.

    Bacchi, Cyrus J; Barker, Robert H; Rodriguez, Aixa; Hirth, Bradford; Rattendi, Donna; Yarlett, Nigel; Hendrick, Clifford L; Sybertz, Edmund


    Genzyme 644131, 8-methyl-5'-{[(Z)-4-aminobut-2-enyl](methylamino)}adenosine, is an analog of the enzyme activated S-adenosylmethionine decarboxylase (AdoMetDC) inhibitor and the trypanocidal agent MDL-7381, 5-{[(Z)-4-aminobut-2-enyl](methylamino)}adenosine. The analog differs from the parent in having an 8-methyl group on the purine ring that bestows favorable pharmacokinetic, biochemical, and trypanocidal activities. The compound was curative in acute Trypanosoma brucei brucei and drug-resistant Trypanosoma brucei rhodesiense model infections, with single-dose activity in the 1- to 5-mg/kg/day daily dose range for 4 days against T. brucei brucei and 25- to 50-mg/kg twice-daily dosing against T. brucei rhodesiense infections. The compound was not curative in the TREU 667 central nervous system model infection but cleared blood parasitemia and extended time to recrudescence in several groups. This study shows that AdoMetDC remains an attractive chemotherapeutic target in African trypanosomes and that chemical changes in AdoMetDC inhibitors can produce more favorable drug characteristics than the lead compound.

  10. Adenosine prevents TNFα-induced decrease in endothelial mitochondrial mass via activation of eNOS-PGC-1α regulatory axis.

    Theodore J Kalogeris

    Full Text Available We tested whether adenosine, a cytoprotective mediator and trigger of preconditioning, could protect endothelial cells from inflammation-induced deficits in mitochondrial biogenesis and function. We examined this question using human microvascular endothelial cells exposed to TNFα. TNFα produced time and dose-dependent decreases in mitochondrial membrane potential, cellular ATP levels, and mitochondrial mass, preceding an increase in apoptosis. These effects were prevented by co-incubation with adenosine, a nitric oxide (NO donor, a guanylate cyclase (GC activator, or a cell-permeant cyclic GMP (cGMP analog. The effects of adenosine were blocked by a nitric oxide synthase inhibitor, a soluble guanylate cyclase inhibitor, a morpholino antisense oligonucleotide to endothelial nitric oxide synthase (eNOS, or siRNA knockdown of the transcriptional coactivator, PGC-1α. Incubation with exogenous NO, a GC activator, or a cGMP analog reversed the effect of eNOS knockdown, while the effect of NO was blocked by inhibition of GC. The protective effects of NO and cGMP analog were prevented by siRNA to PGC-1α. TNFα also decreased expression of eNOS, cellular NO levels, and PGC-1α expression, which were reversed by adenosine. Exogenous NO, but not adenosine, rescued expression of PGC-1α in cells in which eNOS expression was knocked down by eNOS antisense treatment. Thus, TNFα elicits decreases in endothelial mitochondrial function and mass, and an increase in apoptosis. These effects were reversed by adenosine, an effect mediated by eNOS-synthesized NO, acting via soluble guanylate cyclase/cGMP to activate a mitochondrial biogenesis regulatory program under the control of PGC-1α. These results support the existence of an adenosine-triggered, mito-and cytoprotective mechanism dependent upon an eNOS-PGC-1α regulatory pathway, which acts to preserve endothelial mitochondrial function and mass during inflammatory challenge.

  11. A proton-translocating adenosine triphosphatase is associated with the peroxisomal membrane of yeasts

    Douma, A.C.; Veenhuis, M.; Sulter, G.J.; Harder, W.


    The association of an ATPase with the yeast peroxisomal membrane was established by both biochemical and cytochemical procedures. Peroxisomes were purified from protoplast homogenates of the methanol-grown yeast Hansenula polymorpha by differential and sucrose gradient centrifugation. Biochemical an

  12. GABAB and adenosine receptors mediate enhancement of the K+ current, IAHP, by reducing adenylyl cyclase activity in rat CA3 hippocampal neurons.

    Gerber, U; Gähwiler, B H


    1. Gamma-aminobuturic acid-B (GABAB) and adenosine A1 receptors, which are expressed in hippocampal pyramidal cells, are linked to pertussis toxin-sensitive G-proteins known to be coupled negatively to the enzyme adenylyl cyclase. This study investigates the electrophysiological consequences of adenylyl cyclase inhibition in response to stimulation of these receptors. 2. Single-electrode voltage-clamp recordings were obtained from CA3 pyramidal cells in rat hippocampal slice cultures in presence of tetrodotoxin. The calcium-dependent potassium current (IAHP), which is very sensitive to intracellular levels of adenosine 3',5'-cyclic monophosphate (cAMP), was used as an electrophysiological indicator of adenylyl cyclase activity. 3. Application of baclofen (10 microM), a selective agonist at GABAB receptors, or adenosine (50 microM) each resulted in a transient decrease followed by a significant enhancement in the amplitude of evoked IAHP. The initial reduction in amplitude of IAHP probably reflects inadequacies in voltage clamp of electronically distant dendritic sites, due to the shunting caused by concomitant activation of potassium conductance by baclofen/adenosine. Comparable increases in membrane conductance in response to the GABAA agonist, muscimol, caused a similar reduction in IAHP. The enhancement of IAHP is consistent with an inhibition of constitutively active adenylyl cyclase. 4. The receptor mediating the responses to adenosine was identified as belonging to the A1 subtype on the basis of its sensitivity to the selective antagonist 8-cyclopentyl-1,3-dipropylxanthine.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. Impairment of adenosine A3 receptor activity disrupts neutrophil migratory capacity and impacts innate immune function in vivo.

    Butler, Matt; Sanmugalingam, Devika; Burton, Victoria J; Wilson, Tammy; Pearson, Ruth; Watson, Robert P; Smith, Philip; Parkinson, Scott J


    Adenosine possesses potent anti-inflammatory properties which are partly mediated by G(i) -coupled adenosine A3 receptors (A3Rs). A3R agonists have shown clinical benefit in a number of inflammatory conditions although some studies in A3R-deficient mice suggest a pro-inflammatory role. We hypothesised that, in addition to cell signalling effects, A3R compounds might inhibit neutrophil chemotaxis by disrupting the purinergic feedback loop controlling leukocyte migration. Human neutrophil activation triggered rapid upregulation of surface A3R expression which was disrupted by pre-treatment with either agonist (Cl-IB-MECA) or antagonist (MRS1220). Both compounds reduced migration velocity and neutrophil transmigration capacity without impacting the response to chemokines per se. Similar effects were observed in murine neutrophils, while cells from A3R-deficient mice displayed a constitutively impaired migratory phenotype indicating compound-induced desensitisation and genetic ablation had the same functional outcome. In a dextran sodium sulphate-induced colitis model, A3R-deficient mice exhibited reduced colon pathology and decreased tissue myeloperoxidase levels at day 8 - consistent with reduced neutrophil recruitment. However, A3R-deficient mice were unable to resolve the dextran sodium sulphate-induced inflammation and had elevated numbers of tissue-associated bacteria by day 21. Our data indicate that A3Rs play a role in neutrophil migration and disrupting this function has the potential to adversely affect innate immune responses.

  14. Prevention of adenosine A2A receptor activation diminishes beat-to-beat alternation in human atrial myocytes.

    Molina, Cristina E; Llach, Anna; Herraiz-Martínez, Adela; Tarifa, Carmen; Barriga, Montserrat; Wiegerinck, Rob F; Fernandes, Jacqueline; Cabello, Nuria; Vallmitjana, Alex; Benitéz, Raúl; Montiel, José; Cinca, Juan; Hove-Madsen, Leif


    Atrial fibrillation (AF) has been associated with increased spontaneous calcium release from the sarcoplasmic reticulum and linked to increased adenosine A2A receptor (A2AR) expression and activation. Here we tested whether this may favor atrial arrhythmogenesis by promoting beat-to-beat alternation and irregularity. Patch-clamp and confocal calcium imaging was used to measure the beat-to-beat response of the calcium current and transient in human atrial myocytes. Responses were classified as uniform, alternating or irregular and stimulation of Gs-protein coupled receptors decreased the frequency where a uniform response could be maintained from 1.0 ± 0.1 to 0.6 ± 0.1 Hz; p < 0.01 for beta-adrenergic receptors and from 1.4 ± 0.1 to 0.5 ± 0.1 Hz; p < 0.05 for A2ARs. The latter was linked to increased spontaneous calcium release and after-depolarizations. Moreover, A2AR activation increased the fraction of non-uniformly responding cells in HL-1 myocyte cultures from 19 ± 3 to 51 ± 9 %; p < 0.02, and electrical mapping in perfused porcine atria revealed that adenosine induced electrical alternans at longer cycle lengths, doubled the fraction of electrodes showing alternation, and increased the amplitude of alternations. Importantly, protein kinase A inhibition increased the highest frequency where uniform responses could be maintained from 0.84 ± 0.12 to 1.86 ± 0.11 Hz; p < 0.001 and prevention of A2AR-activation with exogenous adenosine deaminase selectively increased the threshold from 0.8 ± 0.1 to 1.2 ± 0.1 Hz; p = 0.001 in myocytes from patients with AF. In conclusion, A2AR-activation promotes beat-to-beat irregularities in the calcium transient in human atrial myocytes, and prevention of A2AR activation may be a novel means to maintain uniform beat-to-beat responses at higher beating frequencies in patients with atrial fibrillation.

  15. A2A adenosine receptor antagonism enhances synaptic and motor effects of cocaine via CB1 cannabinoid receptor activation.

    Alessandro Tozzi

    Full Text Available BACKGROUND: Cocaine increases the level of endogenous dopamine (DA in the striatum by blocking the DA transporter. Endogenous DA modulates glutamatergic inputs to striatal neurons and this modulation influences motor activity. Since D2 DA and A2A-adenosine receptors (A2A-Rs have antagonistic effects on striatal neurons, drugs targeting adenosine receptors such as caffeine-like compounds, could enhance psychomotor stimulant effects of cocaine. In this study, we analyzed the electrophysiological effects of cocaine and A2A-Rs antagonists in striatal slices and the motor effects produced by this pharmacological modulation in rodents. PRINCIPAL FINDINGS: Concomitant administration of cocaine and A2A-Rs antagonists reduced glutamatergic synaptic transmission in striatal spiny neurons while these drugs failed to produce this effect when given in isolation. This inhibitory effect was dependent on the activation of D2-like receptors and the release of endocannabinoids since it was prevented by L-sulpiride and reduced by a CB1 receptor antagonist. Combined application of cocaine and A2A-R antagonists also reduced the firing frequency of striatal cholinergic interneurons suggesting that changes in cholinergic tone might contribute to this synaptic modulation. Finally, A2A-Rs antagonists, in the presence of a sub-threshold dose of cocaine, enhanced locomotion and, in line with the electrophysiological experiments, this enhanced activity required activation of D2-like and CB1 receptors. CONCLUSIONS: The present study provides a possible synaptic mechanism explaining how caffeine-like compounds could enhance psychomotor stimulant effects of cocaine.

  16. Activation of adenosine receptor potentiates the anticonvulsant effect of phenytoin against amygdala kindled seizures.

    Sun, Zhen; Zhong, Xiao-Ling; Zong, Yu; Wu, Zhong-Chen; Zhang, Qun; Yu, Jin-Tai; Tan, Lan


    Drug resistance in epilepsy is considered as a complicated and multifactorial problem. Poor penetration of antiepileptic drugs (AEDs) across blood-brain barrier (BBB) into the brain, which results in insufficient level of the drugs at the targeted brain region, has been discussed as one mechanism contributing to pharmacoresistance of epilepsies. Therefore, modulating permeability of BBB is the effective treatment strategy since it facilitates the entry of AEDs into the central nervous system (CNS). Recently, signaling through receptors for the adenosine has been identified as a potent modulator of BBB permeability. This paper aimed to investigate the effects of auxiliary application of adenosine receptor (AR) agonist on amygdala-kindled seizures in adult male Wistar rats. When fully kindled seizures were achieved by daily electrical stimulation of the amygdala, rats were randomly divided into three groups: control, phenytoin, and phenytoin (PHT)+5'-N-ethylcarboxamidoadenosine (NECA) groups. NECA (0.08 mg/kg, i.v.) was applied to the PHT+NECA group after the administration of PHT (75 mg/kg, i.p. on the first day; 50mg/kg, i.p. on the following 9 days). Intravenous infusion of NECA resulted in a significant increase in brain PHT levels as compared with the PHT treatment alone. On the other hand, the auxiliary application of NECA dramatically decreased the frequency of generalized seizures and seizure stage, shortened duration of afterdischarge and generalized seizures, as well as the elevated the afterdischarge threshold and generalized seizures threshold. Our study demonstrated that auxiliary application of AR agonist enhanced brain antiepileptic drug levels and strengthened the anticonvulsant properties of PHT against amygdala kindled seizures.

  17. Recombinant mouse PAP has pH-dependent ectonucleotidase activity and acts through A(1-adenosine receptors to mediate antinociception.

    Nathaniel A Sowa

    Full Text Available Prostatic acid phosphatase (PAP is expressed in nociceptive neurons and functions as an ectonucleotidase. When injected intraspinally, the secretory isoforms of human and bovine PAP protein have potent and long-lasting antinociceptive effects that are dependent on A(1-adenosine receptor (A(1R activation. In this study, we purified the secretory isoform of mouse (mPAP using the baculovirus expression system to determine if recombinant mPAP also had antinociceptive properties. We found that mPAP dephosphorylated AMP, and to a much lesser extent, ADP at neutral pH (pH 7.0. In contrast, mPAP dephosphorylated all purine nucleotides (AMP, ADP, ATP at an acidic pH (pH 5.6. The transmembrane isoform of mPAP had similar pH-dependent ectonucleotidase activity. A single intraspinal injection of mPAP protein had long-lasting (three day antinociceptive properties, including antihyperalgesic and antiallodynic effects in the Complete Freund's Adjuvant (CFA inflammatory pain model. These antinociceptive effects were transiently blocked by the A(1R antagonist 8-cyclopentyl-1, 3-dipropylxanthine (CPX, suggesting mPAP dephosphorylates nucleotides to adenosine to mediate antinociception just like human and bovine PAP. Our studies indicate that PAP has species-conserved antinociceptive effects and has pH-dependent ectonucleotidase activity. The ability to metabolize nucleotides in a pH-dependent manner could be relevant to conditions like inflammation where tissue acidosis and nucleotide release occur. Lastly, our studies demonstrate that recombinant PAP protein can be used to treat chronic pain in animal models.

  18. The adenosine A2A receptor agonist CGS 21680 exhibits antipsychotic-like activity in Cebus apella monkeys

    Andersen, M B; Fuxe, K; Werge, T;


    The adenosine A2A receptor agonist CGS 21680 has shown effects similar to dopamine antagonists in behavioural assays in rats predictive for antipsychotic activity, without induction of extrapyramidal side-effects (EPS). In the present study, we examined whether this functional dopamine antagonism...... and lack of EPS in rodents could also be observed in non-human primates. We investigated the effects of CGS 21680 on behaviours induced by D-amphetamine and (-)-apomorphine in EPS-sensitized Cebus apella monkeys. CGS 21680 was administered s.c. in doses of 0.01, 0.025 and 0.05 mg/kg, alone...... and in combination with D-amphetamine and (-)-apomorphine. The monkeys were videotaped after drug administration and the tapes were rated for EPS and psychosis-like symptoms. CGS 21680 decreased apomorphine-induced behavioural unrest, arousal (0.01-0.05 mg/kg) and stereotypies (0.05 mg/kg) while amphetamine...

  19. Some neural effects of adenosin.

    Haulică, I; Brănişteanu, D D; Petrescu, G H


    The possible neural effects of adenosine were investigated by using electrophysiological techniques at the level of some central and peripheral synapses. The evoked potentials in the somatosensorial cerebral cortex are influenced according to both the type of administration and the level of the electrical stimulation. While the local application does not induce significant alterations, the intrathalamic injections and the perfusion of the IIIrd cerebral ventricle do change the distribution of activated units at the level of different cortical layers especially during the peripheral stimulation. The frequency of spontaneous miniature discharges intracellularly recorded in the neuromuscular junction (mepp) is significantly depressed by adenosine. This effect is calcium- and dose-dependent. The end plate potentials (EPP) were also depressed. The statistical binomial analysis of the phenomenon indicated that adenosine induces a decrease if the presynaptic pool of the available transmitter. The data obtained demonstrate a presynaptic inhibitory action of adenosine beside its known vascular and metaholic effects.

  20. Rutin restores adenosine deaminase activity in serum and the liver and improves biochemical parameters in streptozotocin-induced diabetic rats



    Full Text Available ABSTRACT denosine deaminase (ADA is a critical control point in the regulation of adenosine levels. This study aimed to investigate the effects of a polyphenolic flavonoid, rutin, on the activity of ADA in serum, the cerebral cortex, liver, kidney, and biochemical parameters in diabetic rats. The animals were divided into four groups (n=6 for the following treatments: control; diabetic (streptozotocin 55 mg/kg; diabetic with rutin (100 mg/kg/day; diabetic with glibenclamide (10 mg/kg/day. After 30 days, ADA activity and biochemical parameters were analyzed. The ADA activity in the serum was significantly elevated in the diabetic group compared to the control group (p<0.01. The treatment with rutin prevented the increase in ADA activity in the STZ-induced rats when compared to control group. Our data showed that rutin reduced glucose, LDL levels, and hepatic enzymes in comparison with the control group. These results demonstrate that the increase of ADA activity observed in diabetic rats may be an important indicator of the immunopathogenesis of hyperglycemic disorders and suggest that rutin is important for regulating the enzymatic activities associated with immune, hyperglycemic, and inflammatory response in diabetes mellitus.

  1. The effects of methylmercury on motor activity are sex- and age-dependent, and modulated by genetic deletion of adenosine receptors and caffeine administration.

    Björklund, Olga; Kahlström, Johan; Salmi, Peter; Ogren, Sven Ove; Vahter, Marie; Chen, Jiang-Fan; Fredholm, Bertil B; Daré, Elisabetta


    Adenosine and its receptors are, as part of the brain stress response, potential targets for neuroprotective drugs. We have investigated if the adenosine receptor system affects the developmental neurotoxicity caused by the fish pollutant methylmercury (MeHg). Behavioral outcomes of low dose perinatal MeHg exposure were studied in mice where the A(1) and A(2A) adenosine receptors were either partially blocked by caffeine treatment or eliminated by genetic modification (A(1)R and A(2A)R knock-out mice). From gestational day 7 to day 7 of lactation dams were administered doses that mimic human intake via normal diet, i.e. 1microM MeHg and/or 0.3g/l caffeine in the drinking water. This exposure to MeHg resulted in a doubling of brain Hg levels in wild type females and males at postnatal day 21 (PND21). Open field analysis was performed at PND21 and 2 months of age. MeHg caused time-dependent behavioral alterations preferentially in male mice. A decreased response to amphetamine in 2-month-old males pointed to disturbances in dopaminergic functions. Maternal caffeine intake induced long-lasting changes in the offspring evidenced by an increased motor activity and a modified response to psychostimulants in adult age, irrespectively of sex. Similar alterations were observed in A(1)R knock-out mice, suggesting that adenosine A(1) receptors are involved in the alterations triggered by caffeine exposure during development. Perinatal caffeine treatment and, to some extent, genetic elimination of adenosine A(1) receptors, attenuated the behavioral consequences of MeHg in males. Importantly, also deletion of the A(2A) adenosine receptor reduced the vulnerability to MeHg, consistent with the neuroprotective effects of adenosine A(2A) receptor inactivation observed in hypoxia and Parkinson's disease. Thus, the consequences of MeHg toxicity during gestation and lactation can be reduced by adenosine A(1) and A(2A) receptor inactivation, either via their genetic deletion or by

  2. An orally active adenosine A1 receptor antagonist, FK838, increases renal excretion and maintains glomerular filtration rate in furosemide-resistant rats

    Schnackenberg, Christine G; Merz, Emily; Brooks, David P


    Loop and thiazide diuretics are common therapeutic agents for the treatment of sodium retention and oedema. However, resistance to diuretics and decreases in renal function can develop during diuretic therapy. Adenosine causes renal vasoconstriction, sodium reabsorption, and participates in the tubuloglomerular feedback mechanism for the regulation of glomerular filtration rate.We tested the hypothesis that the selective adenosine A1 receptor antagonist FK838 is orally active and causes diuresis and natriuresis, but maintains glomerular filtration rate in normal rats or in rats with furosemide resistance.In normal male Sprague – Dawley rats, FK838 dose-dependently increased urine flow and sodium and chloride excretion while sparing potassium. In combination with furosemide, FK838 enhanced the diuretic and natriuretic actions of furosemide to the same extent as hydrochlorothiazide and did not increase the potassium loss in normal rats. In furosemide-resistant rats, FK838 increased urine flow and electrolyte excretion to a greater extent than hydrochlorothiazide. In addition, hydrochlorothiazide significantly decreased glomerular filtration rate, whereas FK838 maintained glomerular filtration rate in furosemide-resistant rats.This study shows that the adenosine A1 receptor antagonist FK838 is orally active and causes potent diuresis and natriuresis and maintains glomerular filtration rate in normal or furosemide-resistant rats. Adenosine A1 receptor antagonists may be novel therapeutics for the treatment of oedema in normal or otherwise diuretic-resistant patients. PMID:12922924

  3. Antihyperlipidemic activity of adenosine triphosphate in rabbits fed a high-fat diet and hyperlipidemic patients.

    Zhang, Lianshan; Liang, Libin; Tong, Tong; Qin, Yuguo; Xu, Yanping; Tong, Xinglong


    Context Recently, adenosine triphosphate (ATP) was occasionally found to decrease the triglyceride (TG) levels in several hyperlipidemic patients in our clinical practice. Objective The study investigates the anti-hyperlipidemic effects of ATP in a high-fat fed rabbit model and hyperlipidemic patients. Materials and methods Twenty-four rabbits were randomly divided into three groups of eight animals each as follows: normal diet, high-fat diet and high-fat diet + ATP group. ATP supplementation (40 mg/day) was started at the 20th day and lasted for 10 days. Serum concentrations of total cholesterol (TC), TG, LDL-C, HDL-C were measured on the 20th day and 30th day. Heart, liver and aorta were subjected histopathological examination. Twenty outpatients diagnosed primary hyperlipidemia took ATP at a dose of 60 mg twice a day for 1 week. Results Feeding rabbits with a high-fat diet resulted in a significant elevation of lipid parameters including TC, TG, LDL-C, VLDL-C compared to the normal diet group (p < 0.01). ATP treatment significantly decreased serum TG level (p < 0.01), whilst other parameters remained statistically unaltered. Meanwhile, ATP significantly reduced the thickness of fat layer in cardiac epicardium (p < 0.05) and pathological gradation of ballooning degeneration in hepatocytes (p < 0.05). After taking ATP for 1 week, hyperlipidemia patients exhibited a significant decrease of TG (p < 0.01), but other lipid parameters had no significant change. Discussion and conclusion The study indicates that ATP selectively decreases serum TG levels in high-fat diet rabbits and hyperlipidemic patients. Therefore, ATP supplementation may provide an effective approach to control TG level.

  4. Serum activities of adenosine deaminase, dipeptidyl peptidase IV and prolyl endopeptidase in patients with fibromyalgia: diagnostic implications.

    Čulić, Ognjen; Cordero, Mario D; Žanić-Grubišić, Tihana; Somborac-Bačura, Anita; Pučar, Lara Batičić; Detel, Dijana; Varljen, Jadranka; Barišić, Karmela


    Fibromyalgia (FM) is a chronic pain syndrome with number of symptoms that present challenge in terms of diagnosis and treatment. Patients with FM show abnormal profile of purines in plasma. In this work, we measured serum activities of enzymes involved in purine metabolism, namely total adenosine deaminase (ADE) and its isoforms (ADE1 and ADE2), ecto-ATPase, and 5'-nucleotidase (5'-NT). We also measured activity of dipeptidyl peptidase IV (DPPIV) and prolyl endopeptidase (PEP). Spectrophotometric and fluorometric methods were used for enzyme activity determinations. Enzyme activities were measured in sera of 24 patients with FM that were not undergoing pharmacological treatment during the study. Control group comprised 32 healthy control subjects. Significantly higher activities of total ADE (P = 0.025) and ADE2 (P = 0.011) were observed in FM patients, while no significant differences in ADE1, ecto-ATPase, and 5'-NT activities (P > 0.05) were found when compared to healthy controls. Moreover, increase in the activity of DPPIV (P = 0.015) and lower activity of PEP (P = 0.011) were also found in the FM group. ROC analysis pointed to different diagnostic sensitivities/specificities for individual enzyme activities measured as follows: ADE (50.0/87.5), ADE2 (41.7/90.6), DPPIV (62.5/71.9), and PEP (83.3/62.5). ADE2 and PEP were shown to be independent predictors of FM, while combination of the two gives AUC of 0.786 (95 % confidence interval of 0.656-0.885, P < 0.05). Our results are showing that serum activities of ADE2 and PEP could be useful as biomarkers for FM diagnosis. However, relatively low diagnostic sensitivity of ADE2 and specificity of PEP must be taken into account.

  5. Methylene blue induces macroautophagy through 5' adenosine monophosphate-activated protein kinase pathway to protect neurons from serum deprivation.

    Xie, Luokun; Li, Wenjun; Winters, Ali; Yuan, Fang; Jin, Kunlin; Yang, Shaohua


    Methylene blue has been shown to be neuroprotective in multiple experimental neurodegenerative disease models. However, the mechanisms underlying the neuroprotective effects have not been fully elucidated. Previous studies have shown that macroautophagy has multiple beneficial roles for maintaining normal cellular homeostasis and that induction of macroautophagy after myocardial ischemia is protective. In the present study we demonstrated that methylene blue could protect HT22 hippocampal cell death induced by serum deprivation, companied by induction of macroautophagy. We also found that methylene blue-mediated neuroprotection was abolished by macroautophagy inhibition. Interestingly, 5' adenosine monophosphate-activated protein kinase (AMPK) signaling, but not inhibition of mammalian target of rapamycin signaling, was activated at 12 and 24 h after methylene blue treatment in a dose-dependent manner. Methylene blue-induced macroautophagy was blocked by AMPK inhibitor. Consistent with in vitro data, macroautophagy was induced in the cortex and hippocampus of mouse brains treated with methylene blue. Our findings suggest that methylene blue-induced neuroprotection is mediated, at least in part, by macroautophagy though activation of AMPK signaling.

  6. Paeoniflorin attenuates neuroinflammation and dopaminergic neurodegeneration in the MPTP model of Parkinson's disease by activation of adenosine A1 receptor.

    Liu, Hua-Qing; Zhang, Wei-Yu; Luo, Xue-Ting; Ye, Yang; Zhu, Xing-Zu


    1. This study examined whether Paeoniflorin (PF), the major active components of Chinese herb Paeoniae alba Radix, has neuroprotective effect in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease (PD). 2. Subcutaneous administration of PF (2.5 and 5 mg kg(-1)) for 11 days could protect tyrosine hydroxylase (TH)-positive substantia nigra neurons and striatal nerve fibers from death and bradykinesia induced by four-dose injection of MPTP (20 mg kg(-1)) on day 8. 3. When given at 1 h after the last dose of MPTP, and then administered once a day for the following 3 days, PF (2.5 and 5 mg kg(-1)) also significantly attenuated the dopaminergic neurodegeneration in a dose-dependent manner. Post-treatment with PF (5 mg kg(-1)) significantly attenuated MPTP-induced proinflammatory gene upregulation and microglial and astrocytic activation. 4. Pretreatment with 0.3 mg kg(-1) 8-cyclopentyl-1,3-dipropylxanthine, an adenosine A1 receptor (A1AR) antagonist, 15 min before each dose of PF, reversed the neuroprotective and antineuroinflammatory effects of PF. 5. In conclusion, this study demonstrated that PF could reduce the MPTP-induced toxicity by inhibition of neuroinflammation by activation of the A1AR, and suggested that PF might be a valuable neuroprotective agent for the treatment of PD.

  7. Mixed inhibition of adenosine deaminase activity by 1,3-dinitrobenzene: a model for understanding cell-selective neurotoxicity in chemically-induced energy deprivation syndromes in brain.

    Wang, Yipei; Liu, Xin; Schneider, Brandon; Zverina, Elaina A; Russ, Kristen; Wijeyesakere, Sanjeeva J; Fierke, Carol A; Richardson, Rudy J; Philbert, Martin A


    Astrocytes are acutely sensitive to 1,3-dinitrobenzene (1,3-DNB) while adjacent neurons are relatively unaffected, consistent with other chemically-induced energy deprivation syndromes. Previous studies have investigated the role of astrocytes in protecting neurons from hypoxia and chemical injury via adenosine release. Adenosine is considered neuroprotective, but it is rapidly removed by extracellular deaminases such as adenosine deaminase (ADA). The present study tested the hypothesis that ADA is inhibited by 1,3-DNB as a substrate mimic, thereby preventing adenosine catabolism. ADA was inhibited by 1,3-DNB with an IC(50) of 284 μM, Hill slope, n = 4.8 ± 0.4. Native gel electrophoresis showed that 1,3-DNB did not denature ADA. Furthermore, adding Triton X-100 (0.01-0.05%, wt/vol), Nonidet P-40 (0.0015-0.0036%, wt/vol), or bovine serum albumin (0.05 mg/ml or changing [ADA] (0.2 and 2 nM) did not substantially alter the 1,3-DNB IC(50) value. Likewise, dynamic light scattering showed no particle formation over a (1,3-DNB) range of 149-1043 μM. Kinetics revealed mixed inhibition with 1,3-DNB binding to ADA (K(I) = 520 ± 100 μM, n = 1 ± 0.6) and the ADA-adenosine complex (K(IS) = 262 ± 7 μM, n = 6 ± 0.6, indicating positive cooperativity). In accord with the kinetics, docking predicted binding of 1,3-DNB to the active site and three peripheral sites. In addition, exposure of DI TNC-1 astrocytes to 10-500 μM 1,3-DNB produced concentration-dependent increases in extracellular adenosine at 24 h. Overall, the results demonstrate that 1,3-DNB is a mixed inhibitor of ADA and may thus lead to increases in extracellular adenosine. The finding may provide insights to guide future work on chemically-induced energy deprivation.

  8. Calcium and adenosine triphosphate control of cellular pathology: asparaginase-induced pancreatitis elicited via protease-activated receptor 2

    Peng, Shuang; Gerasimenko, Julia V.; Tsugorka, Tatiana; Gryshchenko, Oleksiy; Samarasinghe, Sujith; Gerasimenko, Oleg V.


    Exocytotic secretion of digestive enzymes from pancreatic acinar cells is elicited by physiological cytosolic Ca2+ signals, occurring as repetitive short-lasting spikes largely confined to the secretory granule region, that stimulate mitochondrial adenosine triphosphate (ATP) production. By contrast, sustained global cytosolic Ca2+ elevations decrease ATP levels and cause necrosis, leading to the disease acute pancreatitis (AP). Toxic Ca2+ signals can be evoked by products of alcohol and fatty acids as well as bile acids. Here, we have investigated the mechanism by which l-asparaginase evokes AP. Asparaginase is an essential element in the successful treatment of acute lymphoblastic leukaemia, the most common type of cancer affecting children, but AP is a side-effect occurring in about 5–10% of cases. Like other pancreatitis-inducing agents, asparaginase evoked intracellular Ca2+ release followed by Ca2+ entry and also substantially reduced Ca2+ extrusion because of decreased intracellular ATP levels. The toxic Ca2+ signals caused extensive necrosis. The asparaginase-induced pathology depended on protease-activated receptor 2 and its inhibition prevented the toxic Ca2+ signals and necrosis. We tested the effects of inhibiting the Ca2+ release-activated Ca2+ entry by the Ca2+ channel inhibitor GSK-7975A. This markedly reduced asparaginase-induced Ca2+ entry and also protected effectively against the development of necrosis. This article is part of the themed issue ‘Evolution brings Ca2+ and ATP together to control life and death’. PMID:27377732

  9. Guanosine may increase absence epileptic activity by means of A2A adenosine receptors in Wistar Albino Glaxo Rijswijk rats.

    Lakatos, Renáta Krisztina; Dobolyi, Árpád; Todorov, Mihail Ivilinov; Kékesi, Katalin A; Juhász, Gábor; Aleksza, Magdolna; Kovács, Zsolt


    The non-adenosine nucleoside guanosine (Guo) was demonstrated to decrease quinolinic acid(QA)-induced seizures, spontaneously emerged absence epileptic seizures and lipopolysaccharide(LPS)-evoked induction of absence epileptic seizures suggesting its antiepileptic potential. It was also described previously that intraperitoneal (i.p.) injection of 20 and 50mg/kg Guo decreased the number of spike-wave discharges (SWDs) in a well investigated model of human absence epilepsy, the Wistar Albino Glaxo Rijswijk (WAG/Rij) rats during 4th (20mg/kg Guo) and 3rd as well as 4th (50mg/kg Guo) measuring hours. Guanosine can potentially decrease SWD number by means of its putative receptors but absence epileptic activity changing effects of Guo by means of increased extracellular adenosine (Ado) cannot be excluded. An increase in the dose of i.p. injected Guo is limited by its low solubility in saline, therefore, we addressed in the present study whether higher doses of Guo, diluted in sodium hydroxide (NaOH) solution, have more potent antiepileptic effect in WAG/Rij rats. We confirmed that i.p. 50mg/kg Guo decreased but, surprisingly, i.p. 100mg/kg Guo enhanced the number of SWDs in WAG/Rij rats. Combined i.p. injection of a non-selective Ado receptor antagonist theophylline (5mg/kg) or a selective Ado A2A receptor (A2AR) antagonist SCH 58261 (7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine) (1mg/kg) and a cyclooxygenase 1 and 2/COX-1 and COX-2 inhibitor indomethacin (10mg/kg) with 100mg/kg Guo decreased the SWD number compared to i.p. 100mg/kg Guo alone. The results suggest that i.p. 100mg/kg Guo can increase SWD number by means of the adenosinergic system.

  10. Study of the essentiality of the Aspergillus fumigatus triA gene, encoding RNA triphosphatase, using the heterokaryon rescue technique and the conditional gene expression driven by the alcA and niiA promoters.

    Monteiro, M Cândida; De Lucas, J Ramón


    The identification of essential genes represents a critical step in the discovery of novel therapeutic targets in Aspergillus fumigatus. Structural analyses of the Saccharomyces cerevisiae RNA triphosphatase pointed out this enzyme as an attractive therapeutic target for fungal infections. In addition, demonstration of the essentiality of the S. cerevisiae RNA triphosphatase encoding gene enhanced the value of this potential therapeutic target. Nevertheless, consideration of a fungal RNA triphosphatase as an ideal therapeutic target needs confirmation of the essentiality of the respective gene in a fungal pathogen. In this work, we analyzed the essentiality of the A. fumigatus triA gene, encoding RNA triphosphatase, by conditional gene expression and heterokaryon deletion. Using the conditional gene expression driven by the alcA promoter (alcA(P)), we found that TriA depletion causes morphological abnormalities that result in a very strong growth inhibition. Nevertheless, since a strict terminal phenotype was not observed, the essentiality of the triA gene could not be ensured. Accordingly, the essentiality of this gene was analyzed by the heterokaryon rescue technique. Results obtained unequivocally demonstrated the essentiality of the A. fumigatus triA gene, indicating the suitability of the RNA triphosphatase as an ideal therapeutic target to treat A. fumigatus infections. Besides, a second conditional gene expression system, based on the niiA promoter (niiA(P)), was utilized in this work. Although the niiA(P)-mediated repression of triA was less severe than that driven by the alcA(P), a strong growth inhibition was also found in niiA(P)-triA strains. Finally, E-tests performed to determine whether triA down-regulated cells became more sensitive to antifungals suggest a synergic effect between amphotericin B and another antifungal inhibiting the A. fumigatus RNA triphosphatase activity.

  11. Comparison of the Effects of Adenosine A1 Receptors Activity in CA1 Region of the Hippocampus on Entorhinal Cortex and Amygdala Kindled Seizures in Rats

    A. Heidarianpour


    Full Text Available Introduction & Objective: In the CNS, adenosine is known to suppress repetitive neuronal Firing, suggesting a role as an endogenous modifier of seizures. Indeed, intracerebral adenosine concentrations rise acutely during seizure activity and are thought to be responsible for terminating seizures and establishing a period of post-ictal refractoriness. However, it is unclear whether this suppression results from a general depression of brain excitability or through action on particular sites critical for the control of after discharge generation and/or seizure development and propagation. In this regard, comparison of the effects of adenosine A1 receptors of CA1 (region of the ‎hippocampus on entorhinal cortex and amygdala kindled seizures was ‎investigated in this study. Materials & Methods: In this experimental study, Animals were kindled by daily electrical stimulation of amygdale (group A or entorhinal cortex (group B. In the fully kindled animals, N6-‎cyclohexyladenosine (CHA;1 and 10 M; a selective adenosine A1 receptor ‎agonist and 1,3-dimethyl-8-cyclohexylxanthine(CPT;1 ‎µ‎M; a selective ‎adenosine A1 receptors antagonist were microinfused bilaterally into the CA1 ‎region of hippocampus (1l/2min and animals were stimulated at 5 and 15 minutes after drug ‎injection. All animals were received artificial cerebrospinal fluid, 24 h before ‎each drug injection and this result were used as control. Results: The seizure parameters were measured at 5 and 15min post injection. Obtained data showed that CHA at concentrations of 10 ‎µ‎M reduced ‎entorhinal cortex and amygdala after discharge and stage5 seizure durations and ‎increased stage4 latency. CHA at concentration 1‎µ‎M significantly alters ‎seizure parameters of group A but not effect on group B. Intrahippocampal (CA1 region pretreatment of CPT (1 ‎µ‎M before CHA abolished the effects of CHA on seizure parameters.Conclusion: It ‎may be

  12. Adenosine A1 receptor activation modulates N-methyl-d-aspartate (NMDA) preconditioning phenotype in the brain.

    Constantino, Leandra C; Pamplona, Fabrício A; Matheus, Filipe C; Ludka, Fabiana K; Gomez-Soler, Maricel; Ciruela, Francisco; Boeck, Carina R; Prediger, Rui D; Tasca, Carla I


    N-methyl-d-aspartate (NMDA) preconditioning is induced by subtoxic doses of NMDA and it promotes a transient state of resistance against subsequent lethal insults. Interestingly, this mechanism of neuroprotection depends on adenosine A1 receptors (A1R), since blockade of A1R precludes this phenomenon. In this study we evaluated the consequences of NMDA preconditioning on the hippocampal A1R biology (i.e. expression, binding properties and functionality). Accordingly, we measured A1R expression in NMDA preconditioned mice (75mg/kg, i.p.; 24h) and showed that neither the total amount of receptor, nor the A1R levels in the synaptic fraction was altered. In addition, the A1R binding affinity to the antagonist [(3)H] DPCPX was slightly increased in total membrane extracts of hippocampus from preconditioned mice. Next, we evaluated the impact of NMDA preconditioning on A1R functioning by measuring the A1R-mediated regulation of glutamate uptake into hippocampal slices and on behavioral responses in the open field and hot plate tests. NMDA preconditioning increased glutamate uptake into hippocampal slices without altering the expression of glutamate transporter GLT-1. Interestingly, NMDA preconditioning also induced antinociception in the hot plate test and both effects were reversed by post-activation of A1R with the agonist CCPA (0.2mg/kg, i.p.). NMDA preconditioning or A1R modulation did not alter locomotor activity in the open field. Overall, the results described herein provide new evidence that post-activation of A1R modulates NMDA preconditioning-mediated responses, pointing to the importance of the cross-talk between glutamatergic and adenosinergic systems to neuroprotection.

  13. Adenosine A2A receptor activation reduces recurrence and mortality from Clostridium difficile infection in mice following vancomycin treatment

    Li Yuesheng


    Full Text Available Abstract Background Activation of the A2A adenosine receptor (A2AAR decreases production of inflammatory cytokines, prevents C. difficile toxin A-induced enteritis and, in combination with antibiotics, increases survival from sepsis in mice. We investigated whether A2AAR activation improves and A2AAR deletion worsens outcomes in a murine model of C. difficile (strain VPI10463 infection (CDI. Methods C57BL/6 mice were pretreated with an antibiotic cocktail prior to infection and then treated with vancomycin with or without an A2AAR agonist. A2AAR-/- and littermate wild-type (WT mice were similarly infected, and IFNγ and TNFα were measured at peak of and recovery from infection. Results Infected, untreated mice rapidly lost weight, developed diarrhea, and had mortality rates of 50-60%. Infected mice treated with vancomycin had less weight loss and diarrhea during antibiotic treatment but mortality increased to near 100% after discontinuation of antibiotics. Infected mice treated with both vancomycin and an A2AAR agonist, either ATL370 or ATL1222, had minimal weight loss and better long-term survival than mice treated with vancomycin alone. A2AAR KO mice were more susceptible than WT mice to death from CDI. Increases in cecal IFNγ and blood TNFα were pronounced in the absence of A2AARs. Conclusion In a murine model of CDI, vancomycin treatment resulted in reduced weight loss and diarrhea during acute infection, but high recurrence and late-onset death, with overall mortality being worse than untreated infected controls. The administration of vancomycin plus an A2AAR agonist reduced inflammation and improved survival rates, suggesting a possible benefit of A2AAR agonists in the management of CDI to prevent recurrent disease.

  14. Enzymatic activity of granulations tissues under low doses of radiation. Biochemical analysis in rats; Estudo da atividade enzimatica em tecidos de granulacao de ratos submetidos a baixas doses de radiacao

    Tosoni, Guilherme Monteiro [UNESP, Araraquara, SP (Brazil). Faculdade de Odontologia. Dept. de Diagnostico e Cirurgia; Boscolo, Frab Norberto; Cury, Jaime Aparecido [Universidade Estadual de Campinas, Piracicaba, SP (Brazil). Faculdade de Odontologia; Watanabe, Plauto Christopher Aranha [Sao Paulo Univ., Ribeirao Preto, SP (Brazil). Faculdade de Odontologia. Dept. de Estomatologia


    This paper was designed to investigate in the rat subcutaneous sponge-induced granulation tissue under low doses of X-ray, the activity of alkaline phosphatase, 5`nucleotide phosphodiesterase and adenosine triphosphatase (ATPase) enzymes. One hundred and fourteen Wistar rats were divided into three groups, as follows: Group I as control, Group II that received single 7,14 R in split-dosis immediately after sponge-implantation at the third and fifth days postoperatively. Biopsies were taken after 7, 11, 14, 21 and 28 days and the activity of the three enzymes was determined. The results have shown that in Group II alkaline phosphatase had higher activity in the 14th day of tissue evolution when compared to Groups I and III . The 5`nucleotide phosphodiesterase activity in Group I was similar in all days checked, although in Group II the enzyme showed higher activity in 7th day and lower in 21st. In Group III the activity was higher after 14 and 7 days and lower after 28 and 21 days. There was no observation of changing in adenosine triphosphatase (ATPase) activity when the three groups were compared. (author) 28 refs., 3 tabs.

  15. Deletion of striatal adenosine A(2A) receptor spares latent inhibition and prepulse inhibition but impairs active avoidance learning.

    Singer, Philipp; Wei, Catherine J; Chen, Jiang-Fan; Boison, Detlev; Yee, Benjamin K


    Following early clinical leads, the adenosine A(2A)R receptor (A(2A)R) has continued to attract attention as a potential novel target for treating schizophrenia, especially against the negative and cognitive symptoms of the disease because of A(2A)R's unique modulatory action over glutamatergic in addition to dopaminergic signaling. Through (i) the antagonistic interaction with the dopamine D(2) receptor, and (ii) the regulation of glutamate release and N-methyl-d-aspartate receptor function, striatal A(2A)R is ideally positioned to fine-tune the dopamine-glutamate balance, the disturbance of which is implicated in the pathophysiology of schizophrenia. However, the precise function of striatal A(2A)Rs in the regulation of schizophrenia-relevant behavior is poorly understood. Here, we tested the impact of conditional striatum-specific A(2A)R knockout (st-A(2A)R-KO) on latent inhibition (LI) and prepulse inhibition (PPI) - behavior that is tightly regulated by striatal dopamine and glutamate. These are two common cross-species translational tests for the assessment of selective attention and sensorimotor gating deficits reported in schizophrenia patients; and enhanced performance in these tests is associated with antipsychotic drug action. We found that neither LI nor PPI was significantly affected in st-A(2A)R-KO mice, although a deficit in active avoidance learning was identified in these animals. The latter phenotype, however, was not replicated in another form of aversive conditioning - namely, conditioned taste aversion. Hence, the present study shows that neither learned inattention (as measured by LI) nor sensory gating (as indexed by PPI) requires the integrity of striatal A(2A)Rs - a finding that may undermine the hypothesized importance of A(2A)R in the genesis and/or treatment of schizophrenia.

  16. Lipopolysaccharide-induced serotonin transporter up-regulation involves PKG-I and p38MAPK activation partially through A3 adenosine receptor.

    Zhao, Rui; Wang, Shoubao; Huang, Zhonglin; Zhang, Li; Yang, Xiuying; Bai, Xiaoyu; Zhou, Dan; Qin, Zhizhen; Du, Guanhua


    Serotonin transporter (SERT) is a critical determinant of synaptic serotonin (5-hydroxytryptamine, 5-HT) inactivation which plays a critical role in the pathology of depression and other mood disorders. Lipopolysaccharide (LPS), a potent activator of the inflammatory system, has been reported to cause depression symptoms by the modulation of SERT in vivo and in vitro. This study is aimed to investigate the underlying mechanism of LPS-induced SERT modulation. The 4-(4-(dimethylamino) styryl)-N-methylpyridinium iodide (ASP) assay was used to detect dynamic 5-HT uptake as read out of SERT activities in RBL-2H3 cells, and cytosol Ca(2+) concentrations ([Ca(2+)]i) and nitric oxide (NO) were examined. Using specific cyclic GMP-dependent protein kinase type I (PKG-I), p38 mitogen-activated protein kinases (p38MAPK) and A3 adenosine receptor (A3AR) inhibitors, SERT expression was evaluated by western blot and immunofluorescence analysis. Results showed that 24 h treatment with LPS stimulated 5-HT transport and up-regulate plasma membrane distribution of SERT in RBL-2H3 cells. LPS treatment increased NO and [Ca(2+)]i, and led to significant increases in levels of phosphorylated calcium/calmodulin-dependent protein kinase type II (CaMK-II), inducible NOS (iNOS) and PKG-I as well as active p38 MAPK. Moreover, PKG-I inhibitor KT5823 or p38MAPK inhibitor SB203580 respectively impaired SERT activation and transposition to plasma membrane by LPS. Notably, A3 adenosine receptor inhibitor MRS1191 also hindered SERT stimulation by LPS. In conclusion, LPS-induced 5-HT uptake and transposition to plasma membrane of SERT in RBL-2H3 cells involves CaMK-II/iNOS/PKG-I and p38 MAPK activation, which may be partially mediated by A3 adenosine receptor activation. This finding provides a novel insight into the interrelationship between LPS and depression.

  17. AMP is an adenosine A1 receptor agonist.

    Rittiner, Joseph E; Korboukh, Ilia; Hull-Ryde, Emily A; Jin, Jian; Janzen, William P; Frye, Stephen V; Zylka, Mark J


    Numerous receptors for ATP, ADP, and adenosine exist; however, it is currently unknown whether a receptor for the related nucleotide adenosine 5'-monophosphate (AMP) exists. Using a novel cell-based assay to visualize adenosine receptor activation in real time, we found that AMP and a non-hydrolyzable AMP analog (deoxyadenosine 5'-monophosphonate, ACP) directly activated the adenosine A(1) receptor (A(1)R). In contrast, AMP only activated the adenosine A(2B) receptor (A(2B)R) after hydrolysis to adenosine by ecto-5'-nucleotidase (NT5E, CD73) or prostatic acid phosphatase (PAP, ACPP). Adenosine and AMP were equipotent human A(1)R agonists in our real-time assay and in a cAMP accumulation assay. ACP also depressed cAMP levels in mouse cortical neurons through activation of endogenous A(1)R. Non-selective purinergic receptor antagonists (pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid and suramin) did not block adenosine- or AMP-evoked activation. Moreover, mutation of His-251 in the human A(1)R ligand binding pocket reduced AMP potency without affecting adenosine potency. In contrast, mutation of a different binding pocket residue (His-278) eliminated responses to AMP and to adenosine. Taken together, our study indicates that the physiologically relevant nucleotide AMP is a full agonist of A(1)R. In addition, our study suggests that some of the physiological effects of AMP may be direct, and not indirect through ectonucleotidases that hydrolyze this nucleotide to adenosine.

  18. Allosteric activation of brain hexokinase by magnesium ions and by magnesium ion--adenosine triphosphate complex.

    Bachelard, H S


    1. Substrate-saturation curves of brain hexokinase for MgATP(2-) were sigmoidal at sub-saturating concentrations of glucose when the Mg(2+)/ATP ratio was maintained at 1:1. Under identical conditions, except that Mg(2+) was present in excess, hyperbolic curves were observed. 2. The number of binding sites (calculated from Hill plots) is 1.8 at a Mg(2+)/ATP ratio 1:1, and 1.0 with excess of Mg(2+). The apparent K(m) for MgATP(2-) is 6.5x10(-4)m at a Mg(2+)/ATP ratio 1:1, and 3.5x10(-4)m with excess of Mg(2+). 3. Interdependence between substrate-binding sites was indicated by the effects of varying the concentration of glucose. The sigmoidality and deviation from Michaelis-Menten kinetics at a Mg(2+)/ATP ratio 1:1 became less pronounced with increasing glucose concentration. Also, although substrate-saturation curves for glucose were hyperbolic when the Mg(2+)/ATP ratio was 1:1, reciprocal plots were non-linear. These were linear with excess of Mg(2+). 4. High concentrations of Mg(2+) (Mg(2+)/ATP ratios above 5:1) were inhibitory. 5. The results are taken to indicate homotropic co-operative binding of MgATP(2-) and that Mg(2+) is an allosteric activator. Possible implications in regulation are discussed.

  19. Mast cell adenosine receptors function: a focus on the A3 adenosine receptor and inflammation

    Noam eRudich


    Full Text Available Adenosine is a metabolite, which has long been implicated in a variety of inflammatory processes. Inhaled adenosine provokes bronchoconstriction in asthmatics or chronic obstructive pulmonary disease (COPD patients, but not in non-asthmatics. This hyper responsiveness to adenosine appears to be mediated by mast cell activation. These observations have marked the receptor that mediates the bronchoconstrictor effect of adenosine on mast cells, as an attractive drug candidate. Four subtypes (A1, A2a, A2b and A3 of adenosine receptors have been cloned and shown to display distinct tissue distributions and functions. Animal models have firmly established the ultimate role of the A3 adenosine receptor (A3R in mediating hyper responsiveness to adenosine in mast cells, although the influence of the A2b adenosine receptor was confirmed as well. In contrast, studies of the A3R in humans have been controversial. In this review, we summarize data on the role of different adenosine receptors in mast cell regulation of inflammation and pathology, with a focus on the common and distinct functions of the A3R in rodent and human mast cells. The relevance of mouse studies to the human is discussed.

  20. Adenosine A2 receptor activation ameliorates mitochondrial oxidative stress upon reperfusion through the posttranslational modification of NDUFV2 subunit of complex I in the heart.

    Xu, Jingman; Bian, Xiyun; Liu, Yuan; Hong, Lan; Teng, Tianming; Sun, Yuemin; Xu, Zhelong


    While it is well known that adenosine receptor activation protects the heart from ischemia/reperfusion injury, the precise mitochondrial mechanism responsible for the action remains unknown. This study probed the mitochondrial events associated with the cardioprotective effect of 5'-(N-ethylcarboxamido) adenosine (NECA), an adenosine A2 receptor agonist. Isolated rat hearts were subjected to 30min ischemia followed by 10min of reperfusion, whereas H9c2 cells experienced 20min ischemia and 10min reperfusion. NECA prevented mitochondrial structural damage, decreases in respiratory control ratio (RCR), and collapse of mitochondrial membrane potential (ΔΨm). Both the adenosine A2A receptor antagonist SCH58261 and A2B receptor antagonist MRS1706 inhibited the action of NECA. NECA reduced mitochondrial proteins carbonylation, H2O2, and superoxide generation at reperfusion, but did not change superoxide dismutase (SOD) activity. In support, the protective effects of NECA and Peg-SOD on ΔΨm upon reperfusion were additive, implying that NECA's protection is attributable to the reduced superoxide generation but not to the enhancement of the superoxide-scavenging capacity. NECA increased the mitochondrial Src tyrosine kinase activity and suppressed complex I activity at reperfusion in a Src-dependent manner. NECA also reduced mitochondrial superoxide through Src tyrosine kinase. Studies with liquid chromatography-mass spectrometer (LC-MS) identified Tyr118 of the NDUFV2 subunit of complex 1 as a likely site of the tyrosine phosphorylation. Furthermore, the complex I activity of cells transfected with the Y118F mutant was increased, suggesting that this site might be a negative regulator of complex I activity. In support, NECA failed to suppress complex I activity at reperfusion in cells transfected with the Y118F mutant of NDUFV2. In conclusion, NECA prevents mitochondrial oxidative stress by decreasing mitochondrial superoxide generation through inhibition of complex I

  1. A1-adenosine acute withdrawal response and cholecystokinin-8 induced contractures are regulated by Ca(2+)- and ATP-activated K(+) channels.

    Cascio, Maria Grazia; Valeri, Daniela; Tucker, Steven J; Marini, Pietro


    In isolated guinea-pig ileum (GPI), the A1-adenosine acute withdrawal response is under the control of several neuronal signalling systems, including the μ/κ-opioid and the cannabinoid CB1 systems. It is now well established that after the stimulation of the A1-adenosine system, the indirect activation of both μ/κ-opioid and CB1 systems is prevented by the peptide cholecystokinin-8 (CCk-8). In the present study, we have investigated the involvement of the Ca(2+)/ATP-activated K(+) channels in the regulation of both acute A1-withdrawal and CCk-8-induced contractures in the GPI preparation. Interestingly, we found that: (a) the A1-withdrawal contracture is inhibited by voltage dependent Ca(2+)-activated K(+) channels, Kv, while it is enhanced by the voltage independent Ca(2+)-activated K(+) channels, SKCa; (b) in the presence of CCk-8, the inhibitory effect of the A1 agonist, CPA, on the peptide induced contracture is significantly enhanced by the voltage independent Ca(2+)-activated K(+) channel, SKCa; and (c) the A1-withdrawal contracture precipitated in the presence of CCk-8 is controlled by the ATP-sensitive potassium channels, KATP. Our data suggest, for the first time, that both Ca(2+)- and ATP-activated K(+) channels are involved in the regulation of both A1-withdrawal precipitated and CCk-8 induced contractures.

  2. Adenosine: An immune modulator of inflammatory bowel diseases

    Jeff Huaqing Ye; Vazhaikkurichi M Rajendran


    Inflammatory bowel disease (IBD) is a common and lifelong disabling gastrointestinal disease. Emerging treatments are being developed to target inflammatory cytokines which initiate and perpetuate the immune response. Adenosine is an important modulator of inflammation and its anti-inflammatory effects have been well established in humans as well as in animal models. High extracellular adenosine suppresses and resolves chronic inflammation in IBD models. High extracellular adenosine levels could be achieved by enhanced adenosine absorption and increased de novo synthesis. Increased adenosine concentration leads to activation of the A2a receptor on the cell surface of immune and epithelial cells that would be a potential therapeutic target for chronic intestinal inflammation. Adenosine is transported via concentrative nucleoside transporter and equilibrative nucleoside transporter transporters that are localized in apical and basolateral membranes of intestinal epithelial cells, respectively. Increased extracellular adenosine levels activate the A2a receptor, which would reduce cytokines responsible for chronic inflammation.

  3. Sulfate-activating enzymes of Penicillium chrysogenum. The ATP sulfurylase. adenosine 5'-phosphosulfate complex does not serve as a substrate for adenosine 5'-phosphosulfate kinase

    Renosto, F.; Martin, R.L.; Segel, I.H.


    At a noninhibitory steady state concentration of adenosine 5'-phosphosulfate (APS), increasing the concentration of Penicillium chrysogenum ATP sulfurylase drives the rate of the APS kinase-catalyzed reaction toward zero. The result indicates that the ATP sulfurylase.APS complex does not serve as a substrate for APS kinase, i.e. there is no ''substrate channeling'' of APS between the two sulfate-activating enzymes. APS kinase had no effect on the (S)0.5 values, nH values, or maximum isotope trapping in the single turnover of ATP sulfurylase-bound (/sup 35/S)APS. Equimolar APS kinase (+/- MgATP or APS) also had no effect on the rate constants for the inactivation of ATP sulfurylase by phenylglyoxal, diethylpyrocarbonate, or N-ethylmaleimide. Similarly, ATP sulfurylase (+/- ligands) had no effect on the inactivation of equimolar APS kinase by trinitrobenzene sulfonate, diethylpyrocarbonate, or heat. (The last promotes the dissociation of dimeric APS kinase to inactive monomers.) ATP sulfurylase also had no effect on the reassociation of APS kinase subunits at low temperature. The cumulative results suggest that the two sulfate activating enzymes do not associate to form a ''3'-phosphoadenosine 5'-phosphosulfate synthetase'' complex.

  4. Effect of intermittent umbilical cord occlusion on fetal respiratory activity and brain adenosine in late-gestation sheep.

    Watson, Carole S; Schaefer, Rachel; White, Susan E; Homan, Jacobus H; Fraher, Laurence; Harding, Richard; Bocking, Alan D


    It was hypothesized that intermittent umbilical cord occlusion (UCO) would inhibit ovine fetal breathing movements (FBM) in association with increased cerebral adenosine levels. To test this hypothesis, on two successive days during late gestation (133-134 days; term = 146 days), microdialysis samples were collected from the brains of 10 chronically instrumented fetal sheep during 2-h periods of complete UCO induced every 30 min (Day 1: 2-min UCOs; Day 2: 4-min UCOs). Control fetuses (n = 10) underwent no UCO. Tracheal pressure was measured throughout. This regimen resulted in a decrease in fetal arterial PO2 (PaO2) during each UCO to 7.3 +/- 0.8 mmHg (Pfluid (ECF) adenosine during UCO increased by 219 +/- 215% (Pmechanisms.

  5. Hyperthermia-induced seizures alter adenosine A1 and A2A receptors and 5'-nucleotidase activity in rat cerebral cortex.

    León-Navarro, David Agustín; Albasanz, José L; Martín, Mairena


    Febrile seizure is one of the most common convulsive disorders in children. The neuromodulator adenosine exerts anticonvulsant actions through binding adenosine receptors. Here, the impact of hyperthermia-induced seizures on adenosine A1 and A2A receptors and 5'-nucleotidase activity has been studied at different periods in the cerebral cortical area by using radioligand binding, real-time PCR, and 5'-nucleotidase activity assays. Hyperthermic seizures were induced in 13-day-old rats using a warmed air stream from a hair dryer. Neonates exhibited rearing and falling over associated with hindlimb clonus seizures (stage 5 on Racine scale criteria) after hyperthermic induction. A significant increase in A1 receptor density was observed using [(3) H]DPCPX as radioligand, and mRNA coding A1 was observed 48 h after hyperthermia-induced seizures. In contrast, a significant decrease in A2A receptor density was detected, using [(3) H]ZM241385 as radioligand, 48 h after hyperthermia-evoked convulsions. These short-term changes in A1 and A2A receptors were also accompanied by a loss of 5'-nucleotidase activity. No significant variations either in A1 or A2A receptor density or 5'-nucleotidase were observed 5 and 20 days after hyperthermic seizures. Taken together, both regulation of A1 and A2A receptors and loss of 5'-nucleotidase in the cerebral cortex suggest the existence of a neuroprotective mechanism against seizures. Febrile seizure is one of the most common convulsive disorders in children. The consequences of hyperthermia-induced seizures (animal model of febrile seizures) on adenosine A1 and A2A receptors and 5'-nucleotidase activity have been studied at different periods in cerebral cortical area. A significant increase in A1 receptor density and mRNA coding A1 was observed 48 h after hyperthermia-induced seizures. In contrast, a significant decrease in A2A receptor density and 5'-nucleotidase activity was detected 48 h after convulsions evoked by hyperthermia

  6. Adenosine modulation of [Ca2+]i in cerebellar granular cells: multiple adenosine receptors involved.

    Vacas, Javier; Fernández, Mercedes; Ros, Manuel; Blanco, Pablo


    Elimination of adenosine by addition of adenosine deaminase (ADA) to the media leads to alterations in intracellular free calcium concentration ([Ca(2+)](i)) in cerebellar granular cells. Adenosine deaminase brings about increases or decreases in [Ca(2+)](i) depending on the previous activation state of the cell. These effects are dependent on the catalytic activity of adenosine deaminase, since its previous catalytic inactivation with Hg(2+) prevents the above-mentioned changes in intracellular calcium. Extracellular calcium is required for the increase in [Ca(2+)](i) promoted by ADA. This rise is insensitive to thapsigargin, but sensitive to micromolar concentrations of Ni(2+). Toxins specific for L, N and P/Q calcium channels do not overtly reduce this effect. N(6)-Cyclopentyl adenosine (CPA), an A(1) receptor agonist, produces a partial reversion of ADA effects, while CGS21680, A(2A)/A(2B) receptor agonist, slightly enhances them. Expression of A(1), A(2A), A(2B) and A(3) adenosine receptor mRNAs was detected in cerebellar granular cell cultures. These results suggest that adenosine modulate [Ca(2+)](i) in cerebellar granule cells through different adenosine receptor subtypes which, at least in part, seem to act through R-type calcium channels.

  7. Rosuvastatin increases extracellular adenosine formation in humans in vivo: a new perspective on cardiovascular protection.

    Meijer, P; Oyen, W.J.G.; Dekker, D.; Broek, P.H.H. van den; Wouters, C.W.; Boerman, O.C.; Scheffer, G. J.; Smits, P; Rongen, G.A.P.J.M.


    OBJECTIVE: Statins may increase extracellular adenosine formation from adenosine monophosphate by enhancing ecto-5'-nucleotidase activity. This theory was tested in humans using dipyridamole-induced vasodilation as a read-out for local adenosine formation. Dipyridamole inhibits the transport of extracellular adenosine into the cytosol resulting in increased extracellular adenosine and subsequent vasodilation. In addition, we studied the effect of statin therapy in a forearm model of ischemia-...

  8. [Adenosine deaminase in experimental trypanosomiasis: future implications].

    Pérez-Aguilar, Mary Carmen; Rondón-Mercado, Rocío


    The adenosine deaminase represents a control point in the regulation of extracellular adenosine levels, thus playing a critical role in the modulation of purinergic responses to certain pathophysiological events. Several studies have shown that serum and plasma enzyme levels are elevated in some diseases caused by microorganisms, which may represent a compensatory mechanism due to the elevated levels of adenosine and the release of inflammatory mediators. Recent research indicates that adenosine deaminase activity decreases and affects hematological parameters of infected animals with Trypanosoma evansi, so that such alterations could have implications in the pathogenesis of the disease. In addition, the enzyme has been detected in this parasite; allowing the inference that it could be associated with the vital functions of the same, similar to what occurs in mammals. This knowledge may be useful in the association of chemotherapy with specific inhibitors of the enzyme in future studies.

  9. A high isoflavone diet decreases 5' adenosine monophosphate-activated protein kinase activation and does not correct selenium-induced elevations in fasting blood glucose in mice.

    Stallings, Michael T; Cardon, Brandon R; Hardman, Jeremy M; Bliss, Tyler A; Brunson, Scott E; Hart, Chris M; Swiss, Maria D; Hepworth, Squire D; Christensen, Merrill J; Hancock, Chad R


    Selenium (Se) has been implicated as a micronutrient that decreases adenosine monophosphate-activated protein kinase (AMPK) signaling and may increase diabetes risk by reducing insulin sensitivity. Soy isoflavones (IF) are estrogen-like compounds that have been shown to attenuate insulin resistance, hyperglycemia, adiposity, and increased AMPK activation. We hypothesized that a high IF (HIF) diet would prevent the poor metabolic profile associated with high Se intake. The purpose of this study was to examine changes in basal glucose metabolism and AMPK signaling in response to an HIF diet and/or supplemental Se in a mouse model. Male FVB mice were divided into groups receiving either a control diet with minimal IF (low IF) or an HIF diet. Each dietary group was further subdivided into groups receiving either water or Se at a dose of 3 mg Se/kg body weight daily, as Se-methylselenocysteine (SMSC). After 5 months, mice receiving SMSC had elevated fasting glucose (P < .05) and a tendency for glucose intolerance (P = .08). The increase in dietary IF did not result in improved fasting blood glucose. Interestingly, after 6 months, HIF-fed mice had decreased basal AMPK activation in liver and skeletal muscle tissue (P < .05). Basal glucose metabolism was changed by SMSC supplementation as evidenced by increased fasting blood glucose and glucose intolerance. High dietary IF levels did not protect against aberrant blood glucose. In FVB mice, decreased basal AMPK activation is not the mechanism through which Se exerts its effect. These results suggest that more research must be done to elucidate the role of Se and IF in glucose metabolism.




    Full Text Available ABSTRACT: INTRODUCTION: Tuberculosis is major cause of morbidity and mort ality in India as well in other parts of world. It is caused by myc obacterium tuberculosis which primarily affects lung and cause pulmonary tuberculosis. Diag nosis of tuberculosis rests upon a positive history of contact, clinical symptoms, x-ray chest, sputum positivity and AFB culture. Adenosine deaminase (ADA is an enzyme which catalyzes the de amination of adenosine into inosine and ammonia. ADA level is found to be elevated in tuber culosis and typhoid fever where cell mediated immunity is elevated. The ADA level is sig nificantly elevated in tuberculosis and helps to differentiate between tubercular and non tubercu lar diseases. The ADA level is also found to be elevated in serum and pleural fluid in patients of tubercular pleural effusion, tubercular ascitis and tubercular pericardial effusion. METHODS : Routine hemogram, Montoux test, X-ray chest, FNAC of lymph nodes, biopsy of lymph node whene ver required, estimation of serum ADA level and T.B.IgG studies were performed in each cas e. RESULTS: In the present study a total of 45 cases were selected for the study. There are 30 cases of pulmonary tuberculosis and 15 controls. The values of serum ADA and tubercular Ig G in pulmonary tubercular group are significantly higher as compared to those of control s. None of the control for ADA showed significant ratio of positivity (≥1.7. One of the 1 5 cases showed remarkable ratio of positivity (>1.2-1.6 and 14 (93.3% cases showed insignifican t ratio of positivity. Only 2 (13.33% of the 15 cases showed positivity for TB IgG and rest 13 (8 6.66% were regarded negative. CONCLUSIONS: Thus it can be concluded that determination of ser um adenosine deaminase levels can effectively diagnose tuberculosis with s ensitivity of 96.66% and specificity of 93.33% as compared to TBIgG showing sensitivity of 90% and specificity of 86.6%. Also cost of ADA estimation is remarkably

  11. Running out of time: the decline of channel activity and nucleotide activation in adenosine triphosphate-sensitive K-channels

    Proks, Peter; Puljung, Michael C.; Vedovato, Natascia; Sachse, Gregor; Mulvaney, Rachel; Ashcroft, Frances M.


    KATP channels act as key regulators of electrical excitability by coupling metabolic cues—mainly intracellular adenine nucleotide concentrations—to cellular potassium ion efflux. However, their study has been hindered by their rapid loss of activity in excised membrane patches (rundown), and by a second phenomenon, the decline of activation by Mg-nucleotides (DAMN). Degradation of PI(4,5)P2 and other phosphoinositides is the strongest candidate for the molecular cause of rundown. Broad evidence indicates that most other determinants of rundown (e.g. phosphorylation, intracellular calcium, channel mutations that affect rundown) also act by influencing KATP channel regulation by phosphoinositides. Unfortunately, experimental conditions that reproducibly prevent rundown have remained elusive, necessitating post hoc data compensation. Rundown is clearly distinct from DAMN. While the former is associated with pore-forming Kir6.2 subunits, DAMN is generally a slower process involving the regulatory sulfonylurea receptor (SUR) subunits. We speculate that it arises when SUR subunits enter non-physiological conformational states associated with the loss of SUR nucleotide-binding domain dimerization following prolonged exposure to nucleotide-free conditions. This review presents new information on both rundown and DAMN, summarizes our current understanding of these processes and considers their physiological roles. This article is part of the themed issue ‘Evolution brings Ca2+ and ATP together to control life and death’. PMID:27377720

  12. The adenosine A2A receptor agonist CGS 21680 exhibits antipsychotic-like activity in Cebus apella monkeys

    Andersen, M B; Fuxe, K; Werge, T


    and lack of EPS in rodents could also be observed in non-human primates. We investigated the effects of CGS 21680 on behaviours induced by D-amphetamine and (-)-apomorphine in EPS-sensitized Cebus apella monkeys. CGS 21680 was administered s.c. in doses of 0.01, 0.025 and 0.05 mg/kg, alone...... and in combination with D-amphetamine and (-)-apomorphine. The monkeys were videotaped after drug administration and the tapes were rated for EPS and psychosis-like symptoms. CGS 21680 decreased apomorphine-induced behavioural unrest, arousal (0.01-0.05 mg/kg) and stereotypies (0.05 mg/kg) while amphetamine...... showed a functional anti-dopaminergic effect in Cebus apella monkeys without production of EPS. This further substantiates that adenosine A2A receptor agonists may have potential as antipsychotics with atypical profiles....

  13. Attenuation of gastric mucosal inflammation induced by aspirin through activation of A2A adenosine receptor in rats

    Masaru Odashima; Reina Ohba; Sumio Watanabe; Joel Linden; Michiro Otaka; Mario Jin; Koga Komatsu; Isao Wada; Youhei Horikawa; Tamotsu Matsuhashi; Natsumi Hatakeyama; Jinko Oyake


    AIM: To determine whether a specific adenosine A2A receptor agonist (ATL-146e) can ameliorate aspirin-induced gastric mucosal lesions in rats, and reduce neutrophil accumulation and production of pro-inflammatory cytokines.METHODS: Gastric lesions were produced by oral gavage of aspirin (200 mg/kg) and HCl (0.15 mol/L,8.0 mL/kg). 4-{3-[6-Amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-cyclohexanecarboxylic acid methyl ester (ATL-146e,2.5-5 μg/kg, IP) was injected 30 min before the administration of aspirin. Tissue myeloperoxidase (MPO) concentration in gastric mucosa was measured as an index of neutrophil infiltration. Gastric mucosal concentrations of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were determined by ELISA. Also, we examined the effect of ATL-146e on tissue prostaglandin E2 (PGE2) production and gastric secretion.RESULTS: Intragastric administration of aspirin induced multiple hemorrhagic erosions in rat gastric mucosa. The total length of gastric erosions (ulcer index) in control rats was 29.8±7.75 mm and was reduced to 3.8±1.42 mm after pretreatment with 5.0 g/kg ATL-146e (P< 0.01).The gastric contents of MPO and pro-inflammatory cytokines were all increased after the administration of aspirin and reduced to nearly normal levels by ATL-146e.Gastric mucosal PGE2 concentration was not affected by intraperitoneal injection of ATL-146e.CONCLUSION: The specific adenosine A2A receptor agohist, ATL-146e, has potent anti-ulcer effects presumably mediated by its anti-inflammatory properties.

  14. Adenosine monophosphate-activated protein kinase activation enhances embryonic neural stem cell apoptosis in a mouse model of amyotrophic lateral sclerosis

    Yanling Sui; Zichun Zhao; Rong Liu; Bin Cai; Dongsheng Fan


    Alterations in embryonic neural stem cells play crucial roles in the pathogenesis of amyotrophic lateral sclerosis. We hypothesized that embryonic neural stem cells from SOD1G93A individuals might be more susceptible to oxidative injury, resulting in a propensity for neurodegeneration at later stages. In this study, embryonic neural stem cells obtained from human superoxide dis-mutase 1 mutant (SOD1G93A) and wild-type (SOD1WT) mouse models were exposed to H2O2. We assayed cell viability with mitochondrial succinic dehydrogenase colorimetric reagent, and measured cell apoptosis by lfow cytometry. Moreover, we evaluated the expression of the adenos-ine monophosphate-activated protein kinase (AMPK)α-subunit, paired box 3 (Pax3) protein, and p53 in western blot analyses. Compared with SOD1WT cells, SOD1G93A embryonic neural stem cells were more likely to undergo H2O2-induced apoptosis. Phosphorylation of AMPKαin SOD1G93A cells was higher than that in SOD1WT cells. Pax3 expression was inversely correlated with the phosphorylation levels of AMPKα. p53 protein levels were also correlated with AMPKαphosphorylation levels. Compound C, an inhibitor of AMPKα, attenuated the effects of H2O2. These results suggest that embryonic neural stem cells from SOD1G93A mice are more susceptible to apoptosis in the presence of oxidative stress compared with those from wild-type controls, and the effects are mainly mediated by Pax3 and p53 in the AMPKα pathway.

  15. Pretreatment with adenosine and adenosine A1 receptor agonist protects against intestinal ischemia-reperfusion injury in rat

    V Haktan Ozacmak; Hale Sayan


    AIM: To examine the effects of adenosine and A1 receptor activation on reperfusion-induced small intestinal injury.METHODS: Rats were randomized into groups with sham operation, ischemia and reperfusion, and systemic treatments with either adenosine or 2-chloro-N6-cyclopentyladenosine, A1 receptor agonist or 8-cyclopentyl-1,3-dipropylxanthine, A1 receptor antagonist, plus adenosine before ischemia. Following reperfusion, contractions of ileum segments in response to KCl, carbachol and substance P were recorded. Tissue myeloperoxidase,malondialdehyde, and reduced glutathione levels were measured.RESULTS: Ischemia significantly decreased both contraction and reduced glutathione level which were ameliorated by adenosine and agonist administration. Treatment also decreased neutrophil infiltration and membrane lipid peroxidation. Beneficial effects of adenosine were abolished by pretreatment with A1 receptor antagonist.CONCLUSION: The data suggest that adenosine and A1 receptor stimulation attenuate ischemic intestinal injury via decreasing oxidative stress, lowering neutrophil infiltration, and increasing reduced glutathione content.

  16. The Role of Adenosine Signaling in Headache: A Review

    Nathan T. Fried


    Full Text Available Migraine is the third most prevalent disease on the planet, yet our understanding of its mechanisms and pathophysiology is surprisingly incomplete. Recent studies have built upon decades of evidence that adenosine, a purine nucleoside that can act as a neuromodulator, is involved in pain transmission and sensitization. Clinical evidence and rodent studies have suggested that adenosine signaling also plays a critical role in migraine headache. This is further supported by the widespread use of caffeine, an adenosine receptor antagonist, in several headache treatments. In this review, we highlight evidence that supports the involvement of adenosine signaling in different forms of headache, headache triggers, and basic headache physiology. This evidence supports adenosine A2A receptors as a critical adenosine receptor subtype involved in headache pain. Adenosine A2A receptor signaling may contribute to headache via the modulation of intracellular Cyclic adenosine monophosphate (cAMP production or 5' AMP-activated protein kinase (AMPK activity in neurons and glia to affect glutamatergic synaptic transmission within the brainstem. This evidence supports the further study of adenosine signaling in headache and potentially illuminates it as a novel therapeutic target for migraine.

  17. Adenosine stress protocols for myocardial perfusion imaging

    Baškot Branislav


    Full Text Available Background/Aim. Treadmill test combined with myocardial perfusion scintigraphy (MPS is a commonly used technique in the assessment of coronary artery disease. There are many patients, however, who may not be able to undergo treadmill test. Such patients would benefit from pharmacological stress procedures combined with MPS. The most commonly used pharmacological agents for cardiac stress are coronary vasodilatators (adenosine, dipyridamol and catecholamines. Concomitant low-level treadmill exercise with adenosine pharmacologic stress (AdenoEX during MPS has become commonly used in recent years. A number of studies have demonstrated a beneficial impact of AdenoEX protocol. The aim of the study was, besides introducing into practice the two types of protocols of pharmatological stress test with adenosine, as a preparation for MPS, to compare and monitor the frequency of their side effects to quality, acquisition, as well as to standardize the onset time of acquisition (diagnostic imaging for both protocols. Methods. A total of 130 patients underwent pharmacological stress test with adenosine (vasodilatator. In 108 of the patients we performed concomitant exercise (AdenoEX of low level (50W by a bicycle ergometar. In 28 of the patients we performed Adenosine abbreviated protocol (AdenoSCAN. Side effects of adenosine were followed and compared between the two kinds of protocols AdenoEX and AdenoSCAN. Also compared were image quality and suggested time of acquisition after the stress test. Results. Numerous side effects were found, but being short-lived they did not require any active interventions. The benefit of AdenoEX versus AdenoSCAN included decreased side effects (62% vs 87%, improved safety and patients tolerance, improved target-to-background ratios because of less subdiaphragmatic activity, earlier acquisition, and improved sensitivity. Conclusion. The safety and efficacy of adenosine pharmacological stress is even better with concomitant

  18. Brain-derived neurotrophic factor mediates neuroprotection against Aβ-induced toxicity through a mechanism independent on adenosine 2A receptor activation.

    Jerónimo-Santos, André; Fonseca-Gomes, João; Guimarães, Diogo Andrade; Tanqueiro, Sara Ramalho; Ramalho, Rita Mira; Ribeiro, Joaquim Alexandre; Sebastião, Ana Maria; Diógenes, Maria José


    Brain-derived neurotrophic factor (BDNF) promotes neuronal survival through TrkB-FL activation. The activation of adenosine A2A receptors (A2AR) is essential for most of BDNF-mediated synaptic actions, such as synaptic plasticity, transmission and neurotransmitter release. We now aimed at evaluating the A2AR influence upon BDNF-mediated neuroprotection against Aβ25-35 toxicity in cultured neurons. Results showed that BDNF increases cell survival and reduces the caspase-3 and calpain activation induced by amyloid-β (Aβ) peptide, in a mechanism probably dependent on PLCγ pathway. This BDNF-mediated neuroprotection is not affected by A2AR activation or inhibition. Moreover neither activation nor inhibition of A2AR, per se, significantly influenced Aβ-induced neuronal death on calpain-mediated cleavage of TrkB induced by Aβ. In conclusion, these results suggest that, in opposition to the fast synaptic actions of BDNF, the neuroprotective actions of this neurotrophin against a strong Aβ insult do not require the activation of A2AR.

  19. Cows are not mice: the role of cyclic AMP, phosphodiesterases, and adenosine monophosphate-activated protein kinase in the maintenance of meiotic arrest in bovine oocytes.

    Bilodeau-Goeseels, Sylvie


    Meiotic maturation in mammalian oocytes is initiated during fetal development, and is then arrested at the dictyate stage - possibly for several years. Oocyte meiosis resumes in preovulatory follicles in response to the lutenizing hormone (LH) surge or spontaneously when competent oocytes are removed from follicles and cultured. The mechanisms involved in meiotic arrest and resumption in bovine oocytes are not fully understood, and several studies point to important differences between oocytes from rodent and livestock species. This paper reviews earlier and contemporary studies on the effects of cAMP-elevating agents and phosphodiesterase (PDE) enzyme inhibitors on the maintenance of meiotic arrest in bovine oocytes in vitro. Contrary to results obtained with mouse oocytes, bovine oocyte meiosis is inhibited by activators of the energy sensor adenosine monophosphate-activated protein kinase (AMPK, mammalian gene PRKA), which is activated by AMP, the degradation product of cAMP. It is not clear whether or not the effects were due to AMPK activation, and they may depend on culture conditions. Evidence suggests that other signaling pathways (for example, the cGMP/nitric oxide pathway) are involved in bovine oocyte meiotic arrest, but further studies are needed to understand the interactions between the signaling pathways that lead to maturation promoting factor (MPF) being inactive or active. An improved understanding of the mechanisms involved in the control of bovine oocyte meiosis will facilitate better control of the process in vitro, resulting in increased developmental competence and increased efficiency of in vitro embryo production procedures.

  20. Activity of cholinesterases, pyruvate kinase and adenosine deaminase in rats experimentally infected by Fasciola hepatica: Influences of these enzymes on inflammatory response and pathological findings.

    Baldissera, Matheus D; Bottari, Nathieli B; Mendes, Ricardo E; Schwertz, Claiton I; Lucca, Neuber J; Dalenogare, Diessica; Bochi, Guilherme V; Moresco, Rafael N; Morsch, Vera M; Schetinger, Maria R C; Rech, Virginia C; Jaques, Jeandre A; Da Silva, Aleksandro S


    The aim of this study was to investigate acetylcholinesterase (AChE) in total blood and liver tissue; butyrylcholinesterase (BChE) in serum and liver tissue; adenosine deaminase (ADA) in serum and liver tissue; and pyruvate kinase (PK) in liver tissue of rats experimentally infected by Fasciola hepatica. Animals were divided into two groups with 12 animals each, as follows: group A (uninfected) and group B (infected). Samples were collected at 20 (A1 and B1;n=6 each) and 150 (A2 and B2; n=6 each) days post-infection (PI). Infected animals showed an increase in AChE activity in whole blood and a decrease in AChE activity in liver homogenates (P<0.05) at 20 and 150 days PI. BChE and PK activities were decreased (P<0.05) in serum and liver homogenates of infected animals at 150 days PI. ADA activity was decreased in serum at 20 and 150 days PI, while in liver homogenates it was only decreased at 150 days PI (P<0.05). Aspartate aminotransferase and alanine aminotransferase activities in serum were increased (P<0.05), while concentrations of total protein and albumin were decreased (P<0.05) when compared to control. The histological analysis revealed fibrous perihepatitis and necrosis. Therefore, we conclude that the liver fluke is associated with cholinergic and purinergic dysfunctions, which in turn may influence the pathogenesis of the disease.

  1. [Effect of Arnica montana tincture on some hydrolytic enzyme activities of rat liver in experimental toxic hepatitis].

    Iaremiĭ, I M; Meshchyshen, I F; Hrihor'ieva, N P; Kostiuk, L S


    Effects of tinctura arnica on arginase, adenosine triphosphatase, glucose-6-phosphatase and 5'-nucleotidase activities of rats liver in case of experimental toxic hepatitis have been studied. Toxic hepatitis was caused by 2 times interstomach administration of 0.25 ml oil solution of carbon tetrachloride per 100 g of animal weight. 20 mkl/100 g of tinctura arnica was administered every day per os for 14 days. The enzyme activities have been investigated at 3, 7 and 17 days. A significant demention of a studied hydrolytic enzyme activities in rats liver at intoxication of the body by CCI4 has been shown. It has been established that tinctura arnica administered per os to intoxicated animals sped up the normalization of hydrolytic enzyme activities in rat liver.

  2. Mechanism of protection of adenosine from sulphate radical anion and repair of adenosine radicals by caffeic acid in aqueous solution

    M Sudha Swaraga; L Charitha; M Adinarayana


    The photooxidation of adenosine in presence of peroxydisulphate (PDS) has been studied by spectrophotometrically measuring the absorbance of adenosine at 260 nm. The rates of oxidation of adenosine by sulphate radical anion have been determined in the presence of different concentrations of caffeic acid. Increase in [caffeic acid] is found to decrease the rate of oxidation of adenosine suggesting that caffeic acid acts as an efficient scavenger of $SO_{4}^{\\bullet-}$ and protects adenosine from it. Sulphate radical anion competes for adenosine as well as for caffeic acid. The quantum yields of photooxidation of adenosine have been calculated from the rates of oxidation of adenosine and the light intensity absorbed by PDS at 254 nm, the wavelength at which PDS is activated to sulphate radical anion. From the results of experimentally determined quantum yields (exptl) and the quantum yields calculated (cal) assuming caffeic acid acting only as a scavenger of $SO_{4}^{\\bullet-}$ show that exptl values are lower than cal values. The ' values, which are experimentally found quantum yield values at each caffeic acid concentration and corrected for $SO_{4}^{\\bullet-}$ scavenging by caffeic acid, are also found to be greater than exptl values. These observations suggest that the transient adenosine radicals are repaired by caffeic acid in addition to scavenging of sulphate radical anions.

  3. Adenosine and sleep

    Yanik, G.M. Jr.


    Behavioral and biochemical approaches have been used to determine the relative contribution of endogenous adenosine and adenosine receptors to the sleep-wake cycle in the rat. Adenosine concentrations in specific areas of the rat brain were not affected by 24 hours of total sleep deprivation, or by 24 or 48 hours of REM sleep deprivation. In order to assess the effect of REM sleep deprivation on adenosine A/sub 1/ receptors, /sup 3/H-L-PIA binding was measured. The Bmax values for /sup 3/H-L-PIA binding to membrane preparations of the cortices and corpus striata from 48 hour REM sleep-deprived animals were increased 14.8% and 23%, respectively. These increases were not maintained following the cessation of sleep deprivation and recovered within 2 hours. The results of a 96 hour REM deprivation experiment were similar to those of the 48 hour REM sleep deprivation experiment. However, these increases were not evident in similar structures taken from stress control animals, and conclusively demonstrated that the changes in /sup 3/H-L-PIA binding resulted from REM sleep deprivation and not from stress.

  4. Metabolism. AMP-activated protein kinase mediates mitochondrial fission in response to energy stress.

    Toyama, Erin Quan; Herzig, Sébastien; Courchet, Julien; Lewis, Tommy L; Losón, Oliver C; Hellberg, Kristina; Young, Nathan P; Chen, Hsiuchen; Polleux, Franck; Chan, David C; Shaw, Reuben J


    Mitochondria undergo fragmentation in response to electron transport chain (ETC) poisons and mitochondrial DNA-linked disease mutations, yet how these stimuli mechanistically connect to the mitochondrial fission and fusion machinery is poorly understood. We found that the energy-sensing adenosine monophosphate (AMP)-activated protein kinase (AMPK) is genetically required for cells to undergo rapid mitochondrial fragmentation after treatment with ETC inhibitors. Moreover, direct pharmacological activation of AMPK was sufficient to rapidly promote mitochondrial fragmentation even in the absence of mitochondrial stress. A screen for substrates of AMPK identified mitochondrial fission factor (MFF), a mitochondrial outer-membrane receptor for DRP1, the cytoplasmic guanosine triphosphatase that catalyzes mitochondrial fission. Nonphosphorylatable and phosphomimetic alleles of the AMPK sites in MFF revealed that it is a key effector of AMPK-mediated mitochondrial fission.

  5. Comorbidities in Neurology: Is Adenosine the Common Link?

    Boison, Detlev; Aronica, Eleonora


    Comorbidities in Neurology represent a major conceptual and therapeutic challenge. For example, temporal lobe epilepsy (TLE) is a syndrome comprised of epileptic seizures and comorbid symptoms including memory and psychiatric impairment, depression, and sleep dysfunction. Similarly, Alzheimer’s disease (AD), Parkinson’s disease (PD), and Amyotrophic Lateral Sclerosis (ALS) are accompanied by various degrees of memory dysfunction. Patients with AD have an increased likelihood for seizures, whereas all four conditions share certain aspects of psychosis, depression, and sleep dysfunction. This remarkable overlap suggests common pathophysiological mechanisms, which include synaptic dysfunction and synaptotoxicity, as well as glial activation and astrogliosis. Astrogliosis is linked to synapse function via the tripartite synapse, but astrocytes also control the availability of gliotransmitters and adenosine. Here we will specifically focus on the ‘adenosine hypothesis of comorbidities’ implying that astrocyte activation, via overexpression of adenosine kinase (ADK), induces a deficiency in the homeostatic tone of adenosine. We present evidence from patient-derived samples showing astrogliosis and overexpression of ADK as common pathological hallmark of epilepsy, AD, PD, and ALS. We discuss a transgenic ‘comorbidity model’, in which brain-wide overexpression of ADK and resulting adenosine deficiency produces a comorbid spectrum of seizures, altered dopaminergic function, attentional impairment, and deficits in cognitive domains and sleep regulation. We conclude that dysfunction of adenosine signaling is common in neurological conditions, that adenosine dysfunction can explain comorbid phenotypes, and that therapeutic adenosine augmentation might be effective for the treatment of comorbid symptoms in multiple neurological conditions. PMID:25979489

  6. Rhythm generation by the pre-Bötzinger Complex in medullary slice and island preparations: Effects of adenosine A1 receptor activation

    Shields Edward J


    Full Text Available Abstract Background The pre-Bötzinger complex (preBötC is a central pattern generator within the ventrolateral medulla oblongata's ventral respiratory group that is important for the generation of respiratory rhythm. Activation of adenosine A1 receptors (A1R depresses preBötC rhythmogenesis. Although it remains unclear whether A1R activation is important for organisms in a normal metabolic state, A1R activation is important to the response of the preBötC to metabolic stress, such as hypoxia. This study examined mechanisms linking A1R activation to depression of preBötC rhythmogenesis in medullary slice and island preparations from neonatal mice. Results Converting medullary slices to islands by cutting away much of the medullary tissue adjacent to the preBötC decreased the amplitude of action potential bursts generated by a population of neurons within the preBötC (recorded with an extracellular electrode, and integrated using a hardware integrator, without noticeably affecting burst frequency. The A1R agonist N6-Cyclopentyladenosine (NCPA reduced population burst frequency in slices by ca. 33% and in islands by ca. 30%. As in normal (drug-free artificial cerebrospinal fluid (aCSF, NCPA decreased burst frequency in slices when GABAAergic or GABAAergic and glycinergic transmission were blocked, and in islands when GABAAergic transmission was antagonized. Converting slices to island preparations decreased synaptic input to inspiratory neurons. NCPA further decreased the frequency of synaptic inputs to neurons in island preparations and lowered the input resistance of inspiratory neurons, even when chemical communication between neurons and other cells was impeded. Conclusion Together these data support the suggestion that depression of preBötC activity by A1R activation involves both decreased neuronal excitability and diminished inter-neuronal communication.

  7. Involvement of adenosine monophosphate-activated protein kinase in the influence of timed high-fat evening diet on the hepatic clock and lipogenic gene expression in mice.

    Huang, Yan; Zhu, Zengyan; Xie, Meilin; Xue, Jie


    A high-fat diet may result in changes in hepatic clock gene expression, but potential mechanisms are not yet elucidated. Adenosine monophosphate-activated protein kinase (AMPK) is a serine/threonine protein kinase that is recognized as a key regulator of energy metabolism and certain clock genes. Therefore, we hypothesized that AMPK may be involved in the alteration of hepatic clock gene expression under a high-fat environment. This study aimed to examine the effects of timed high-fat evening diet on the activity of hepatic AMPK, clock genes, and lipogenic genes. Mice with hyperlipidemic fatty livers were induced by orally administering high-fat milk via gavage every evening (19:00-20:00) for 6 weeks. Results showed that timed high-fat diet in the evening not only decreased the hepatic AMPK protein expression and activity but also disturbed its circadian rhythm. Accordingly, the hepatic clock genes, including clock, brain-muscle-Arnt-like 1, cryptochrome 2, and period 2, exhibited prominent changes in their expression rhythms and/or amplitudes. The diurnal rhythms of the messenger RNA expression of peroxisome proliferator-activated receptorα, acetyl-CoA carboxylase 1α, and carnitine palmitoyltransferase 1 were also disrupted; the amplitude of peroxisome proliferator-activated receptorγcoactivator 1α was significantly decreased at 3 time points, and fatty liver was observed. These findings demonstrate that timed high-fat diet at night can change hepatic AMPK protein levels, activity, and circadian rhythm, which may subsequently alter the circadian expression of several hepatic clock genes and finally result in the disorder of hepatic lipogenic gene expression and the formation of fatty liver.

  8. Globular adiponectin protects human umbilical vein endothelial cells against apoptosis through adiponectin receptor 1/adenosine monophosphate-activated protein kinase pathway

    ZHAO Hong-yu; ZHAO Min; YI Tong-ning; ZHANG Jin


    Background Endothelial dysfunction is a key event in the onset and progression of atherosclerosis in diabetic patients.Apoptosis may lead to endothelial dysfunction and contribute to vascular complications. However, no study has addressed apoptosis in human umbilical vein endothelial cells (HUVECs) induced by an intermittent high-glucose media and its association with adiponectin receptor 1 (adipoR1), adipoR2, or adenosine monophosphate (AMP)-activated protein kinase (AMPK).Methods HUVECs were cultured in continuous normal glucose (5.5 mmol/L), continuous high glucose (25 mmol/L),alternating normal and high glucose and mannitol. In the alternating normal and high-glucose media, HUVECs were treated under different conditions. First, cells were transfected with the adipoR1-specific small-interfering RNA (siRNA)and then stimulated with globular adiponectin (gAD). Second, cells were cultured in both gAD and the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR). Third, cells were cultured in the AMPK inhibitor adenine-9-β-D-arabino-furanoside (araA), gAD, and in AICAR.Results HUVEC apoptosis increased more significantly in an intermittent high-glucose medium than in a constant high-glucose medium. HUVEC apoptosis induced by an intermittent high-glucose medium was inhibited when the cells were pretreated with 3 μg/ml gAD, which rapidly activated AMPK and adipoR1 in HUVECs. However, adipoR2 was not activated.Conclusions We found that adipoR1, not adipoR2, is involved in mediating intermittent high-concentration glucoseevoked apoptosis in endothelial cells. gAD activated AMPK through adipoR1, leads to the partial inhibition of HUVEC apoptosis. A fluctuating glucose medium is more harmful than a constant high-glucose medium to endothelial cells.

  9. Expression of adenosine 5'-monophosphate-Activated protein kinase (AMPK) in ovine testis (Ovis aries): In vivo regulation by nutritional state.

    Taibi, N; Dupont, J; Bouguermouh, Z; Froment, P; Ramé, C; Anane, A; Amirat, Z; Khammar, F


    In the present study, we identified AMPK and investigated its potential role in steroidogenesis in vivo in the ovine testis in response to variation in nutritional status (fed control vs. restricted). We performed immunoblotting to show that both active and non-active forms of AMPK exist in ovine testis and liver. In testis, we confirmed these results by immunohistochemistry. We found a correlation between ATP (Adenosine-Triphosphate) levels and the expression of AMPK in liver. Also, low and high caloric diets induce isoform-dependent AMPK expression, with an increase in α2, ß1ß2 and γ1 activity levels. Although the restricted group exhibited an increase in lipid balance, only the triglyceride and HC-VLDL (Cholesterol-Very low density lipoprotein) fractions showed significant differences between groups, suggesting an adaptive mechanism. Moreover, the relatively low rate of non-esterified fatty acid released into the circulation implies re-esterification to compensate for the physiological need. In the fed control group, AMPK activates the production of testosterone in Leydig cells; this is, in turn, associated with an increase in the expression of 3ß-HSD (3 beta hydroxy steroid deshydrogenase), p450scc (Cholesterol side-chain cleavage enzyme) and StAR (Steroidogenic acute regulatory protein) proteins induced by decreased MAPK ERK½ (Extracellular signal-regulated kinase -Mitogen-activated protein kinase) phosphorylation. In contrast, in the restricted group, testosterone secretion was reduced but intracellular cholesterol concentration was not. Furthermore, the combination of high levels of lipoproteins and emergence of the p38 MAP kinase pathway suggest the involvement of pro-inflammatory cytokines, as confirmed by transcriptional repression of the StAR protein. Taken together, these results suggest that AMPK expression is tissue dependent.

  10. Temporal variations of adenosine metabolism in human blood.

    Chagoya de Sánchez, V; Hernández-Muñoz, R; Suárez, J; Vidrio, S; Yáñez, L; Aguilar-Roblero, R; Oksenberg, A; Vega-González, A; Villalobos, L; Rosenthal, L; Fernández-Cancino, F; Drucker-Colín, R; Díaz-Muñoz, M


    Eight diurnally active (06:00-23:00 h) subjects were adapted for 2 days to the room conditions where the experiments were performed. Blood sampling for adenosine metabolites and metabolizing enzymes was done hourly during the activity span and every 30 min during sleep. The results showed that adenosine and its catabolites (inosine, hypoxanthine, and uric acid), adenosine synthesizing (S-adenosylhomocysteine hydrolase and 5'-nucleotidase), degrading (adenosine deaminase) and nucleotide-forming (adenosine kinase) enzymes as well as adenine nucleotides (AMP, ADP, and ATP) undergo statistically significant fluctuations (ANOVA) during the 24 h. However, energy charge was invariable. Glucose and lactate chronograms were determined as metabolic indicators. The same data analyzed by the chi-square periodogram and Fourier series indicated ultradian oscillatory periods for all the metabolites and enzymatic activities determined, and 24-h oscillatory components for inosine, hypoxanthine, adenine nucleotides, glucose, and the activities of SAH-hydrolase, 5'-nucleotidase, and adenosine kinase. The single cosinor method showed significant oscillatory components exclusively for lactate. As a whole, these results suggest that adenosine metabolism may play a role as a biological oscillator coordinating and/or modulating the energy homeostasis and physiological status of erythrocytes in vivo and could be an important factor in the distribution of purine rings for the rest of the organism.

  11. Adenosine Diphosphate Ribosylation Factor-GTPaseActivating Protein Stimulates the Transport of AUX1Endosome, Which Relies on Actin Cytoskeletal Organization in Rice Root DevelopmentF

    Cheng Du; Yunyuan XU; Yingdian Wang; Kang Chong


    Polar auxin transport,which depends on polarized subcellular distribution of AUXIN RESISTANT 1/LIKE AUX1 (AUX1/LAX) influx carriers and PIN-FORMED (PIN) efflux carriers,mediates various processes of plant growth and development.Endosomal recycling of PIN1 is mediated by an adenosine diphosphate (ADP)ribosylation factor (ARF)-GTPase exchange factor protein,GNOM.However,the mediation of auxin influx carrier recycling is poorly understood.Here,we report that overexpression of OsAGAP,an ARF-GTPase-activating protein in rice,stimulates vesicle transport from the plasma membrane to the Golgi apparatus in protoplasts and transgenic plants and induces the accumulation of early endosomes and AUX1.AUX1 endosomes could partially colocalize with FM4-64 labeled early endosome after actin disruption.Furthermore,OsAGAP is involved in actin cytoskeletal organization,and its overexpression tends to reduce the thickness and bundling of actin filaments.Fluorescence recovery after photobleaching analysis revealed exocytosis of the AUX1 recycling endosome was not affected in the OsAGAP overexpression cells,and was only slightly promoted when the actin filaments were completely disrupted by Lat B.Thus,we propose that AUX1 accumulation in the OsAGAP overexpression and actin disrupted cells may be due to the fact that endocytosis of the auxin influx carrier AUX1 early endosome was greatly promoted by actin cytoskeleton disruption.

  12. Adenosine Monophosphate-Activated Protein Kinase Abates Hyperglycaemia-Induced Neuronal Injury in Experimental Models of Diabetic Neuropathy: Effects on Mitochondrial Biogenesis, Autophagy and Neuroinflammation.

    Yerra, Veera Ganesh; Kumar, Ashutosh


    Impaired adenosine monophosphate kinase (AMPK) signalling under hyperglycaemic conditions is known to cause mitochondrial dysfunction in diabetic sensory neurons. Facilitation of AMPK signalling is previously reported to ameliorate inflammation and induce autophagic response in various complications related to diabetes. The present study assesses the role of AMPK activation on mitochondrial biogenesis, autophagy and neuroinflammation in experimental diabetic neuropathy (DN) using an AMPK activator (A769662). A769662 (15 and 30 mg/kg, i.p) was administered to Sprague-Dawley rats (250-270 g) for 2 weeks after 6 weeks of streptozotocin (STZ) injection (55 mg/kg, i.p.). Behavioural parameters (mechanical/thermal hyperalgesia) and functional characteristics (motor/sensory nerve conduction velocities (MNCV and SNCV) and sciatic nerve blood flow (NBF)) were assessed. For in vitro studies, Neuro2a (N2A) cells were incubated with 25 mM glucose to simulate high glucose condition and then studied for mitochondrial dysfunction and protein expression changes. STZ administration resulted in significant hyperglycaemia (>250 mg/dl) in rats. A769662 treatment significantly improved mechanical/thermal hyperalgesia threshold and enhanced MNCV, SNCV and NBF in diabetic animals. A769662 exposure normalised the mitochondrial superoxide production, membrane depolarisation and markedly increased neurite outgrowth of N2A cells. Further, AMPK activation also abolished the NF-κB-mediated neuroinflammation. A769662 treatment increased Thr-172 phosphorylation of AMPK results in stimulated PGC-1α-directed mitochondrial biogenesis and autophagy induction. Our study supports that compromised AMPK signalling in hyperglycaemic conditions causes defective mitochondrial biogenesis ultimately leading to neuronal dysfunction and associated deficits in DN and activation of AMPK can be developed as an attractive therapeutic strategy for the management of DN.

  13. Value of adenosine deaminase activity to diagnosing tuberculous pleural effusion and malignant effusion%腺苷脱氨酶诊断结核性和恶性胸腔积液价值

    唐学义; 林香花; 朱敏


    Objective To investigate the clinical value of pleural effusion adenosine deaminase activity to differentiating tuberculous pleural effusion from malignant effusion.Methods A total of 80 inpatients with pleural effusion received thoracoscopy and biopsy of pleura and were diagnosed tuberculous pleuritis (42 cases) and malignant pleural effusion (38 cases).The pleural effusion and adenosine deaminase activity were detected in these two groups.The optimal cutoff for tuberculous pleural effusion was determined with the ROC curve.Results The pleural effusion adenosine deaminase activity and the ratio of pleural fluid to serum adenosine deaminase were (48±25)u/L and 4.2±3.0 in tuberculous pleural effusion patients, significantly higher than those in malignant pleural effusion patients (20±9)u/L and 1.7±1.0 respectively( P<0.01).The serum adenosine deaminase activities were (15±6)u/L and (12±5)u/L in tuberculous pleuritis patients and malignant pleural effusion,which showed no significant difference(P>0.05).The cutoff value of pleural adenosine deaminase determined with ROC curve was 30.7 u/L, the sensitivity was 70.5% and the specificity was 92.2%.Conclusion The pleural effusion adenosine deaminase activity but not the serum adenosine deaminase activity can be used to differentiate tuberculous from malignant pleural effusion.%目的:探讨胸腔积液和血清中腺苷脱氨酶鉴别诊断结核性胸膜炎及恶性胸腔积液的价值.方法:因胸腔积液住院,经胸腔镜检查和胸膜活检病理确诊为结核性胸腹炎患者(结核组)42例和恶性胸腔积液患者(恶性组)38例,分别检测2组胸腔积液及血清中腺苷脱氨酶活性,应用ROC曲线确定结核性胸膜炎患者胸腔积液腺苷脱氨酶的最佳临界值.结果:结核组胸腔积液腺苷脱氨酶活性、胸腔积液腺苷脱氨酶与血清腺苷脱氨酶比值分别为(48±25)u/L和4.2±3.0,高于恶性组的(20±9)u/L和1.7±1.0.差异均有统计学意义(P<0

  14. Caffeine inhibits the activation of hepatic stellate cells induced by acetaldehyde via adenosine A2A receptor mediated by the cAMP/PKA/SRC/ERK1/2/P38 MAPK signal pathway.

    He Wang

    Full Text Available Hepatic stellate cell (HSC activation is an essential event during alcoholic liver fibrosis. Evidence suggests that adenosine aggravates liver fibrosis via the adenosine A2A receptor (A2AR. Caffeine, which is being widely consumed during daily life, inhibits the action of adenosine. In this study, we attempted to validate the hypothesis that caffeine influences acetaldehyde-induced HSC activation by acting on A2AR. Acetaldehyde at 50, 100, 200, and 400 μM significantly increased HSC-T6 cells proliferation, and cell proliferation reached a maximum at 48 h after exposure to 200 μM acetaldehyde. Caffeine and the A2AR antagonist ZM241385 decreased the cell viability and inhibited the expression of procollagen type I and type III in acetaldehyde-induced HSC-T6 cells. In addition, the inhibitory effect of caffeine on the expression of procollagen type I was regulated by A2AR-mediated signal pathway involving cAMP, PKA, SRC, and ERK1/2. Interestingly, caffeine's inhibitory effect on the expression of procollagen type III may depend upon the A2AR-mediated P38 MAPK-dependent pathway.Caffeine significantly inhibited acetaldehyde-induced HSC-T6 cells activation by distinct A2AR mediated signal pathway via inhibition of cAMP-PKA-SRC-ERK1/2 for procollagen type I and via P38 MAPK for procollagen type III.

  15. Histamine H3 receptor activation counteracts adenosine A2A receptor-mediated enhancement of depolarization-evoked [3H]-GABA release from rat globus pallidus synaptosomes.

    Morales-Figueroa, Guadalupe-Elide; Márquez-Gómez, Ricardo; González-Pantoja, Raúl; Escamilla-Sánchez, Juan; Arias-Montaño, José-Antonio


    High levels of histamine H3 receptors (H3Rs) are found in the globus pallidus (GP), a neuronal nucleus in the basal ganglia involved in the control of motor behavior. By using rat GP isolated nerve terminals (synaptosomes), we studied whether H3R activation modified the previously reported enhancing action of adenosine A2A receptor (A2AR) stimulation on depolarization-evoked [(3)H]-GABA release. At 3 and 10 nM, the A2AR agonist CGS-21680 enhanced [(3)H]-GABA release induced by high K(+) (20 mM) and the effect of 3 nM CGS-21680 was prevented by the A2AR antagonist ZM-241385 (100 nM). The presence of presynaptic H3Rs was confirmed by the specific binding of N-α-[methyl-(3)H]-histamine to membranes from GP synaptosomes (maximum binding, Bmax, 1327 ± 79 fmol/mg protein; dissociation constant, Kd, 0.74 nM), which was inhibited by the H3R ligands immepip, clobenpropit, and A-331440 (inhibition constants, Ki, 0.28, 8.53, and 316 nM, respectively). Perfusion of synaptosomes with the H3R agonist immepip (100 nM) had no effect on K(+)-evoked [(3)H]-GABA release, but inhibited the stimulatory action of A2AR activation. In turn, the effect of immepip was blocked by the H3R antagonist clobenpropit, which had no significant effect of its own on K(+)-induced [(3)H]-GABA release. These data indicate that H3R activation selectively counteracts the facilitatory action of A2AR stimulation on GABA release from striato-pallidal projections.

  16. Effects of chronic digitalization on cardiac and renal Na+ + K+-dependent adenosine triphosphate activity and circulating catecholamines in the dog.

    Nechay, B R; Jackson, R E; Ziegler, M G; Neldon, S L; Thompson, J D


    To extend our understanding of the mechanism of action of digitalis drugs, we studied electrocardiograms (ECGs), renal function, plasma concentrations of catecholamines, and myocardial and renal Na+ + K+-dependent adenosine triphosphate (Na+ + K+ ATPase) activity in chronically digitalized dogs. Five healthy, male, mongrel dogs received a therapeutic regimen of digoxin (0.1 mg/kg on day 1 in three divided doses followed by 0.025 mg/kg per day) orally for 2-4 months. This resulted in plasma digoxin concentrations of 1.1 to 4.7 ng/ml as determined by radioimmunoassay. Six control dogs received daily gelatin capsules by mouth. ECGs monitored throughout the study showed no changes. Digitalized dogs had elevated plasma norepinephrine concentrations (347 vs. 137 pg/ml in controls) and no change in plasma epinephrine concentrations. Digitalized dogs had elevated glomerular filtration rates (0.74 vs. 0.94 ml/min per g of kidney) without significant changes in renal handling of electrolytes and water. All of the above studies were done without the aid of restraining drugs or infusions. The animals were killed with an overdose of pentobarbital for in vitro studies. In digitalized dogs, microsomal Na+ + K+ ATPase-specific activity was 26 to 33% lower in the renal cortex, medulla, and papilla, and 46% lower in the cardiac left ventricle than in control dogs. Digitalization did not alter the osmolalities of renal tissues. We conclude that chronic reduction Na+ + K+ ATPase activity by one-third dose does not cause abnormalities in renal handling of electrolytes and water, and inhibition of Na+ + K+ ATPase in the left ventricular muscle by one-half is associated with no obvious ECG changes in the dog. Further, elevated plasma norepinephrine concentrations may contribute to both the therapeutic and the toxic effects of digitalis.

  17. Latent N-methyl-D-aspartate receptors in the recurrent excitatory pathway between hippocampal CA1 pyramidal neurons: Ca(2+)-dependent activation by blocking A1 adenosine receptors.

    Klishin, A; Tsintsadze, T; Lozovaya, N; Krishtal, O


    When performed at increased external [Ca2+]/[Mg2+] ratio (2.5 mM/0.5 mM), temporary block of A1 adenosine receptors in hippocampus [by 8-cyclopentyltheophylline (CPT)] leads to a dramatic and irreversible change in the excitatory postsynaptic current (EPSC) evoked by Schaffer collateral/commissural (SCC) stimulation and recorded by in situ patch clamp in CA1 pyramidal neurons. The duration of the EPSC becomes stimulus dependent, increasing with increase in stimulus strength. The later occurring component of the EPSC is carried through N-methyl-D-aspartate (NMDA) receptor-operated channels but disappears under either the NMDA antagonist 2-amino-5-phosphonovaleric acid (APV) or the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). These findings indicate that the late component of the SCC-evoked EPSC is polysynaptic: predominantly non-NMDA receptor-mediated SCC inputs excite CA1 neurons that recurrently excite each other by predominantly NDMA receptor-mediated synapses. These recurrent connections are normally silent but become active after CPT treatment, leading to enhancement of the late component of the EPSC. The activity of these connections is maintained for at least 2 hr after CPT removal. When all functional NMDA receptors are blocked by dizocilpine maleate (MK-801), subsequent application of CPT leads to a partial reappearance of NMDA receptor-mediated EPSCs evoked by SCC stimulation, indicating that latent NMDA receptors are recruited. Altogether, these findings indicate the existence of a powerful system of NMDA receptor-mediated synaptic contacts in SCC input to hippocampal CA1 pyramidal neurons and probably also in reciprocal connections between these neurons, which in the usual preparation are kept latent by activity of A1 receptors. PMID:8618915

  18. Adenosine improves cardiomyocyte respiratory efficiency.

    Babsky, A M; Doliba, M M; Doliba, N M; Osbakken, M D


    The role of adenosine on the regulation of mitochondrial function has been studied. In order to evaluate this the following experiments were done in isolated rat cardiomyocites and mitochondria using polarographic techniques. Cardiomyocyte oxygen consumption (MVO2) and mitochondrial respiratory function (State 3 and State 4, respiratory control index, and ADP/O ratio) were evaluated after exposure to adenosine. Cardiomyocyte MVO2 was significantly lower in cells previously exposed to adenosine (10 microM, 15 min or 30 min cell incubation) than in cells not exposed to adenosine (control). Addition of dipyridamole (10 microM) or 8-(p-Sulfophenyl) theophylline (50 microM) to cardiomyocytes before adenosine incubation prevented the adenosine-induced changes in MVO2. Mitochondria obtained from isolated perfused beating heart previously perfused with adenosine (10 microM, 30 min heart perfusion) also resulted in significant increases in ADP/O and respiratory control index compared to matching control. Mitochondria isolated from cardiomyocytes previously exposed to adenosine (10 microM, 15 min or 30 min cell incubation) resulted in a significant increase in mitochondrial ADP/O ratio compared to control. Adenosine-induced decrease in cardiomyocyte MVO2 may be related to an increase in efficiency of mitochondrial oxidative phosphorylation, and more economical use of oxygen, which is necessary for survival under ischemic stress.

  19. [The involvement of adenosine and adenosine deaminase in experimental myocardial infarct].

    Stratone, A; Busuioc, A; Roşca, V; Bazgan, L; Popa, M; Hăulică, I


    By the ligature of the left coronary artery in the rat anesthetized with nembutal (10 mg/100 i.p.) a significant increase of the 5'-nucleotidase activity (Wooton method) was noticed 10 minutes after the left ventricle infarction (from an average value of 1038.5 +/- 187 mU/g tissue to 1537 +/- 225 mU/g fresh tissue). The adenosine desaminase levels spectrophotometrically determined by Denstedt technique, do not appear significantly modified 10 or 30 minutes after the left ventricle infarction. The chromatographically determined adenosine levels, by HPLC technique, decrease from the average value of 11.63 +/- 1.4 micrograms/mg PT to 8.60 +/- 1.0 micrograms/mg PT 30 minutes after infarction. The observed changes are explained by the conditions of hypoxia in the infarcted ventricle which lead to the raise in adenosine levels by activating the 5'-nucleotidase and their depression by a very fast metabolism of the same substance.

  20. Comparison of Activities and Properties of Pyrophosphate and Adenosine Triphosphate-Dependent Phosphofructokinases of Black Gram (Phaseolus mungo) Seeds.

    Ashihara, H; Stupavska, S


    Both pyrophosphate-dependent phosphofructokinase (PPi-PFKase, EC and ATPdependent phosphofructokinase (ATP-PFKase, EC 2.7. 1.11) were present in dry and germinated black gram seeds. In the absence of fructose-2,6-biphosphate (F2,6BP), the activity of PPi-PFKase expressed as nmol · min(-1) · (pair of cotyledons)(-1) was much lower than that of ATP-PFKase in both dry and germinated seeds. However, PPi-PFKase was activated by F2,6BP and its activity reached the same level as ATP-PFKase activity. ATP-PFKase showed sigmoidal kinetics respective to fructose-6-phosphate (F6P), while PPi-PFKase exhibited hyperbolic kinetics in the presence of F2,6BP. The F6P concentration for half maximal activity of ATP-PFKase (1.5 mM) was nearly 5 times lower than that of PPi-PFKase (7.1 mM). The apparent Km values of PPi-PFKase for PPi and that of ATP-PFKase for ATP were 0.29 mM and 0.23 mM, respectively. Phosphoenolpyruvate (PEP) and citrate inhibited ATP-PFKase activity, but they did not affect PPi-PFKase activity. The activity of PPi-PFKase was inhibited by Pi, while only a little Pi inhibition was observed in the case of ATP-PFKase. These results suggest that the control mechanism of PPi-PFKase and that of ATP-PFKase are quite different. In contrast to pineapple leaves (Carnal, N. W. and C. C. Black, Biochem. Biophys. Res. Commun. 86, 20-26, 1979) and caster bean seedlings (Krugar et al., FEBS Lett. 153, 409-412, 1983), PPi-PFKase is not the predominant PFKase activity in black gram seeds.

  1. The role of adenosine in Alzheimer's disease.

    Rahman, Anisur


    Alzheimer's disease (AD) is a neurodegenerative disorder of the central nervous system manifested by cognitive and memory deterioration, a variety of neuropsychiatric symptoms, behavioral disturbances, and progressive impairment of daily life activities. Current pharmacotherapies are restricted to symptomatic interventions but do not prevent progressive neuronal degeneration. Therefore, new therapeutic strategies are needed to intervene with these progressive pathological processes. In the past several years adenosine, a ubiquitously released purine ribonucleoside, has become important for its neuromodulating capability and its emerging positive experimental effects in neurodegenerative diseases. Recent research suggests that adenosine receptors play important roles in the modulation of cognitive function. The present paper attempts to review published reports and data from different studies showing the evidence of a relationship between adenosinergic function and AD-related cognitive deficits. Epidemiological studies have found an association between coffee (a nonselective adenosine receptor antagonist) consumption and improved cognitive function in AD patients and in the elderly. Long-term administration of caffeine in transgenic animal models showed a reduced amyloid burden in brain with better cognitive performance. Antagonists of adenosine A2A receptors mimic these beneficial effects of caffeine on cognitive function. Neuronal cell cultures with amyloid beta in the presence of an A2A receptor antagonist completely prevented amyloid beta-induced neurotoxicity. These findings suggest that the adenosinergic system constitutes a new therapeutic target for AD, and caffeine and A2A receptor antagonists may have promise to manage cognitive dysfunction in AD.

  2. Role of adenosine 5'-monophosphate-activated protein kinase in interleukin-6 release from isolated mouse skeletal muscle

    Glund, Stephan; Treebak, Jonas Thue; Long, Yun Chau;


    IL-6 is released from skeletal muscle during exercise and has consequently been implicated to mediate beneficial effects on whole-body metabolism. Using 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR), a pharmacological activator of 5'-AMP-activated protein kinase (AMPK), we tested...... the hypothesis that AMPK modulates IL-6 release from isolated muscle. Skeletal muscle from AMPKalpha2 kinase-dead transgenic, AMPKalpha1 knockout (KO) and AMPKgamma3 KO mice and respective wild-type littermates was incubated in vitro, in the absence or presence of 2 mmol/liter AICAR. Skeletal muscle from wild......-type mice was also incubated with the AMPK activator A-769662. Incubation of mouse glycolytic extensor digitorum longus and oxidative soleus muscle for 2 h was associated with profound IL-6 mRNA production and protein release, which was suppressed by AICAR (P

  3. Apoptotic effects of extract from Cnidium monnieri (L.) Cusson by adenosine monosphosphate-activated protein kinase-independent pathway in HCT116 colon cancer cells.

    Lim, Eun Gyeong; Kim, Guen Tae; Lee, Se Hee; Kim, Sang-Yong; Kim, Young Min


    Colon cancer, a common malignancy, can occur due to poor eating habits and increasing age. Consequently, careful regulation of eating habits may serve as a possible method for preventing the occurrence or progression of colon cancer. Extracts of the fruit of Cnidium monnieri (L.) Cusson are well‑known as an effective herbal medicine for the treatment of pain in female genitalia and carbuncle. However, there have been no studies on the apoptotic effects of Cnidium monnieri (L.) Cusson (CME). Adenosine monophosphate‑activated protein kinase (AMPK), the major regulator of energy metabolism, is activated by metabolic stress, including hypoxia and glucose deprivation. Activation of AMPK inhibits cell proliferation and induces apoptosis through the inhibition of phosphorylated (p)‑Akt and control of B‑cell lymphoma 2 (Bcl‑2) family members. The pro‑apoptotic proteins Bcl‑2‑associated X protein (Bax) and Bcl‑2‑homologous antagonist killer (Bak), are activated by their translocation to mitochondria from the cytosol. Translocation of Bax/Bak induces outer membrane permeabilization and is likely to lead to apoptosis through cytochrome C release and caspase activity. In the present study, the apoptotic effects and influence on mitochondria‑mediated apoptotic proteins of CME in HCT116 cells were assessed. We hypothesized that CME may have an effect on the inhibition of p‑Akt in an AMPK‑independent pathway. The present study demonstrated that CME induced the release of LDH and apoptosis through its inhibition of p‑Akt to control Bcl‑2 and activate Bax and Bak. Co‑treatment with CME and AMPK inhibitors showed that CME‑induced apoptosis does not occurr through a AMPK‑dependent pathway. Therefore, the present study determined, for the first time, that CME induced apoptosis as a result of causing metabolic stresses due to directly regulation of the de‑phosphorylation of Akt, whereas it did not control the AMPK-dependent pathway in HCT116

  4. Possible mechanism of adenosine protection in carbon tetrachloride acute hepatotoxicity. Role of adenosine by-products and glutathione peroxidase.

    Chagoya de Sánchez, V; Hernández-Muñoz, R; Yáñez, L; Vidrio, S; Díaz-Muñoz, M


    Adenosine proved to be an effective hepatoprotector increasing the survival rate of rats receiving lethal doses of CCl4. Searching for the mechanism of action, we found that adenosine transiently prevents the necrotic liver damage associated to an acute CCl4 treatment. The antilipoperoxidative action of the nucleoside was evidenced by a decrease of TBA-reactive products and the diene conjugates elicited by the hepatotoxin. Adenosine's protective effect was demonstrated by reverting the decrease of cytochrome P-450 while preserved intact the activity of the microsomal enzyme glucose-6-phosphatase. CCl4 promoted an increase in the oxidant stress through an enhancement in oxidized glutathione levels. This action was also completely counteracted by the nucleoside. Adenosine was unable to prevent CCl4 activation and, even, increased .CCl3 formation in the presence of PBN in vivo. However, in the presence of the nucleoside, irreversible binding of 14CCl4 to the microsomal lipid fraction of the treated animals was decreased. These results suggest that adenosine protective action might be exerted at the level of the propagation reaction following CCl4 activation. Two possible mechanisms were associated to the nucleoside protection: (1) the peroxide-metabolyzed enzymes, GSH-per, showed a marked increase after 30 minutes of adenosine treatment, which was potentiated by the hepatotoxin, suggesting an important role of this enzyme in the nucleoside's action; (2) the adenosine catabolism induced an increase in uric acid level, and allopurinol, a purine metabolism inhibitor, prevented such elevation as well as the antilipoperoxidative action of adenosine and the increase of GSH-per associated with the nucleoside treatment. These facts strongly suggest that the protective effect elicited by adenosine is not a direct one, but rather is related to its catabolic products, such as uric acid, which has been recognized as a free radical scavenger.

  5. Role of adenosine 5'-monophosphate-activated protein kinase subunits in skeletal muscle mammalian target of rapamycin signaling

    Deshmukh, Atul S.; Treebak, Jonas Thue; Long, Yun Chau


    AMP-activated protein kinase (AMPK) is an important energy-sensing protein in skeletal muscle. Mammalian target of rapamycin (mTOR) mediates translation initiation and protein synthesis through ribosomal S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). AMPK...... activation reduces muscle protein synthesis by down-regulating mTOR signaling, whereas insulin mediates mTOR signaling via Akt activation. We hypothesized that AMPK-mediated inhibitory effects on mTOR signaling depend on catalytic alpha2 and regulatory gamma3 subunits. Extensor digitorum longus muscle from...... in extensor digitorum longus muscle from either alpha2 or gamma3 AMPK KO mice, indicating functional alpha2 and gamma3 subunits of AMPK are required for the reduction in mTOR signaling. AICAR alone was without effect on basal phosphorylation of S6K1 (Thr389), ribosomal protein S6 (Ser235/236), and 4E-BP1 (Thr...

  6. Adenosine deaminase activity is a sensitive marker for the diagnosis of tuberculous pleuritis in patients with very low CD4 counts.

    Kamaldeen Baba

    Full Text Available BACKGROUND: Adenosine Deaminase Activity (ADA is a commonly used marker for the diagnosis of tuberculous pleural effusion. There has been concern about its usefulness in immunocompromised patients, especially HIV positive patients with very low CD4 counts. The objective of this study was to evaluate the sensitivity of ADA in pleural fluid in patients with low CD4 counts. MATERIALS AND METHODS: This was a retrospective case control study. Medical files of patients with tuberculous pleuritis and non-tuberculous pleuritis were reviewed. Clinical characteristics, CD4 cell counts in blood and biochemical markers in pleural fluid, including ADA were recorded. RESULTS: One ninety seven tuberculous pleuritis and 40 non-tuberculous pleuritis patients were evaluated. Using the cut-off value of 30 U/L, the overall sensitivity, specificity, positive likelihood ratio, and negative likelihood ratio of ADA was 94%, 95%, 19, and 0.06 respectively. The mean CD4 cell counts among TB pleuritis patients was 29 and 153 cells/microL in patients with CD4 50 cells/microL, (p0.5. There was no correlation between ADA values and CD4 cell counts (r = -0.120, p = 0.369. CONCLUSION: ADA analysis is a sensitive marker of tuberculous pleuritis even in HIV patients with very low CD4 counts in a high TB endemic region. The ADA assay is inexpensive, rapid, and simple to perform and is of great value for the immediate diagnosis of tuberculous pleuritis while waiting for culture result and this has a positive impact on patient outcome.

  7. New ribosome-inactivating proteins with polynucleotide:adenosine glycosidase and antiviral activities from Basella rubra L. and bougainvillea spectabilis Willd.

    Bolognesi, A; Polito, L; Olivieri, F; Valbonesi, P; Barbieri, L; Battelli, M G; Carusi, M V; Benvenuto, E; Del Vecchio Blanco, F; Di Maro, A; Parente, A; Di Loreto, M; Stirpe, F


    New single-chain (type 1) ribosome-inactivating proteins (RIPs) were isolated from the seeds of Basella rubra L. (two proteins) and from the leaves of Bougainvillea spectabilis Willd. (one protein). These RIPs inhibit protein synthesis both in a cell-free system, with an IC50 (concentration causing 50% inhibition) in the 10(-10) M range, and by various cell lines, with IC50S in the 10(-8)-10(-6) M range. All three RIPs released adenine not only from rat liver ribosomes but also from Escherichia coli rRNA, polyadenylic acid, herring sperm DNA, and artichoke mottled crinkle virus (AMCV) genomic RNA, thus being polynucleotide:adenosine glycosidases. The proteins from Basella rubra had toxicity to mice similar to that of most type 1 RIPs (Barbieri et al., 1993, Biochim Biophys Acta 1154: 237-282) with an LD50 (concentration that is 50% lethal) Bougainvillea spectabilis had an LD50 > 32 The N-terminal sequence of the two RIPs from Basella rubra had 80-93% identity, whereas it differed from the sequence of the RIP from Bougainvillea spectabilis. When tested with antibodies against various RIPs, the RIPs from Basella gave some cross-reactivity with sera against dianthin 32, and weak cross-reactivity with momordin I and momorcochin-S, whilst the RIP from Bougainvillea did not cross-react with any antiserum tested. An RIP from Basella rubra and one from Bougainvillea spectabilis were tested for antiviral activity, and both inhibited infection of Nicotiana benthamiana by AMCV.

  8. Adenosine monophosphate-activated protein kinase from the mud crab, Scylla paramamosain: cDNA cloning and profiles under cold stress



    Adenosine monophosphate-activated protein kinase (AMPK), an important energy sensor, is crucial for organism survival under adverse conditions. In this study, the roles of this gene under cold stress in a warm-water mud crab, Scylla paramamosain was investigated. The full-length cDNA (SpAMPK) was 1884 bp and its open reading frame of 1566 bp was isolated and characterized. The expressions of SpAMPK detected by quantitative real-time PCR (qRT-PCR) in various tissues revealed that the highest expression was in the hepatopancreas. The profiles of SpAMPK gene in the hepatopancreas, chela muscleand gill were detected when the subadult crabs were exposed to the four temperature conditions of 10, 15, 20 and 25◦C. The results showed that the expression patterns of SpAMPK mRNA in the three tissues were significantly higher when crabs were exposed to 15◦C than the other three temperature treatments, while at 10◦C treatment, the SpAMPK mRNA was lowestamong the four temperature treatments. These findings suggested that the high expression of SpAMPK mRNA might initiate ATP-producing pathways to generate energy to cope with cold stress at 15◦C treatment, which was slightly below the range of optimum temperatures; while treatment at 10◦C, far lower than optima, the low expression of SpAMPK mRNA could reduce the energy expenditure and thus induce the crabs into cold anesthesia. The results of SpAMPK in this study might contribute to the understanding of the molecular mechanism of acclimation to cold hardiness in S. paramamosain.

  9. Ablation of adenosine monophosphate-activated protein kinaseα1 in vascular smooth muscle cells promotes diet-induced atherosclerotic calcification in vivo

    CAI Zhe-jun; DING Ye; ZHANG Miao; LU Qiu-lun; WU Sheng-nan; ZHU Huai-ping; SONG Ping; ZOU Ming-hui


    AIM:Atherosclerotic calcification is highly linked with plaque instability and cardiovascular events .Adenosine monophosphate-activated protein kinase ( AMPK) has been involved in the pathogenesis of various cardiovascular disease .The contributions of AMPKαsubunits to the development of atherosclerotic calcification in vivo remained unknown .We hypothesized that AMPKαsubunits may play a role in the development of atherosclerotic calcification .METHODS: Atherosclerotic calcification was generated by 24-week fed of western diet in ApoE-/-background mice .Calcification was evaluated in aortic roots and innominate arteries of ApoE-/-mice or in mice with dual deficiencies of ApoE and AMPKαsubunits globally ( AMPKα1 and AMPKα2 ) , or vascular smooth muscle cell ( VSMC)-specific or macrophage-specific knockout of AMPKα1 with atherosclerotic calcification pone diet . The mechanism of AMPKα1 in regulating Runx2 was further explored in human aortic VSMC .RESULTS: Ablation of AMPKα1 but not AMPKα2 in ApoE-/-background promoted atherosclerotic calcification with increased Runt -related transcription factor ( Runx2 ) expression in VSMC compared with ApoE-/-mice.Conversely, chronic administration of metformin, which activated AMPK, markedly reduced ath-erosclerotic calcification and Runx2 expression in ApoE-/-mice but had less effects in ApoE-/-/AMPKα1 -/-mice.Furthermore, VSMC-but not macrophage-specific deficiency of AMPKα1 in ApoE-/-background promoted atherosclerotic calcification in vivo com-pared with the controls .AMPKα1 silencing in human aortic VSMC prevented Runx 2 from proteasome degradation to trigger osteoblastic differentiation of VSMC .Conversely , activation of AMPK led to Runx 2 instability by inducing its small ubiquitin-like modifier modifi-cation (SUMOylation).Protein inhibitor of activated STAT-1 (PIAS1), the SUMO E3-ligase of Runx2, was directly phosphorylated by AMPKα1 at serine 510, to enhance its SUMO E3-ligase activity.Ablation of PIAS1

  10. Vasoconstrictor and vasodilator effects of adenosine in the kidney

    Hansen, Pernille B; Schnermann, Jurgen


    Adenosine is an ATP breakdown product that in most vessels causes vasodilatation and that contributes to the metabolic control of organ perfusion, i.e., to the match between oxygen demand and oxygen delivery. In the renal vasculature, in contrast, adenosine can produce vasoconstriction, a respons...... activation from changes in vascular adenosine concentration, a characteristic that is ideally suited for the role of renal adenosine as a paracrine factor in the control of glomerular function.......Adenosine is an ATP breakdown product that in most vessels causes vasodilatation and that contributes to the metabolic control of organ perfusion, i.e., to the match between oxygen demand and oxygen delivery. In the renal vasculature, in contrast, adenosine can produce vasoconstriction, a response...... that has been suggested to be an organ-specific version of metabolic control designed to restrict organ perfusion when transport work increases. However, the vasoconstriction elicited by an intravenous infusion of adenosine is only short lasting, being replaced within 1-2 min by vasodilatation. It appears...

  11. An improved red blood cell additive solution maintains 2,3-diphosphoglycerate and adenosine triphosphate levels by an enhancing effect on phosphofructokinase activity during cold storage

    P. Burger; H. Korsten; D. de Korte; E. Rombout; R. van Bruggen; A.J. Verhoeven


    BACKGROUND: Current additive solutions (ASs) for red blood cells (RBCs) do not maintain constant 2,3-diphosphoglycerate (DPG) and adenosine triphosphate (ATP) levels during cold storage We have previously shown that with a new AS called phosphate-adenine-glucose-guanosine-gluconate-mannitol (PAGGGM)

  12. Effects of adenosine agonist R-phenylisopropyl-adenosine on halothane anesthesia and antinociception in rats

    Hai-chun MA; Yan-fen WANG; Chun-sheng FENG; Hua ZHAO; Shuji DOHI


    Aim: To investigate the antinociceptive effect of adenosine agonist Rphenylisopropyl-adenosine (R-PIA) given to conscious rats by intracerebroventricular (ICV) and intrathecal (IT), and identify the effect of R-PIA on minimum alveolar concentration (MAC) of halothane with pretreatment of A1 receptor an tagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) or K+ channel blocker 4-aminopyridine (4-AP). Methods: Sprague-Dawley rats were implanted with 24 gauge stainless steel guide cannula using stereotaxic apparatus and ICV method, and an IT catheter (PE-10, 8.5 cm) was inserted into the lumbar subarachnoid space, while the rats were under pentobarbital anesthesia. After one week of recovery from surgery, rats were randomly assigned to one of the following protocols: MAC of halothane, or tail-flick latency. All measurements were performed after R-PIA (0.8-2.0 μg) microinjection into ICV and IT with or without pretreatment of DPCPX or 4-AP. Results: Microinjection of adenosine agonist R PIA in doses of 0.8-2.0 μg into ICV and IT produced a significant dose- and time dependent antinociceptive action as reflected by increasing latency times and ICV administration of adenosine agonist R-PIA (0.8 μg) reducing halothane anes thetic requirements (by 29%). The antinociception and reducing halothane requirements effected by adenosine agonist R-PIA was abolished by DPCPX and 4-AP. Conclusion: ICV and IT administration of adenosine agonist R-PIA produced an antinociceptive effect in a dose-dependent manner and decreased hal othane MAC with painful stimulation through activation of A1 receptor subtype, and the underlying mechanism involves K+ channel activation.

  13. Paeoniflorin attenuates neuroinflammation and dopaminergic neurodegeneration in the MPTP model of Parkinson's disease by activation of adenosine A1 receptor

    Liu, Hua-Qing; Zhang, Wei-Yu; Luo, Xue-Ting; Ye, Yang; Zhu, Xing-Zu


    This study examined whether Paeoniflorin (PF), the major active components of Chinese herb Paeoniae alba Radix, has neuroprotective effect in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease (PD).Subcutaneous administration of PF (2.5 and 5 mg kg−1) for 11 days could protect tyrosine hydroxylase (TH)-positive substantia nigra neurons and striatal nerve fibers from death and bradykinesia induced by four-dose injection of MPTP (20 mg kg−1) on day 8.When...

  14. Fast-scan Cyclic Voltammetry for the Characterization of Rapid Adenosine Release.

    Nguyen, Michael D; Venton, B Jill


    Adenosine is a signaling molecule and downstream product of ATP that acts as a neuromodulator. Adenosine regulates physiological processes, such as neurotransmission and blood flow, on a time scale of minutes to hours. Recent developments in electrochemical techniques, including fast-scan cyclic voltammetry (FSCV), have allowed direct detection of adenosine with sub-second temporal resolution. FSCV studies have revealed a novel mode of rapid signaling that lasts only a few seconds. This rapid release of adenosine can be evoked by electrical or mechanical stimulations or it can be observed spontaneously without stimulation. Adenosine signaling on this time scale is activity dependent; however, the mode of release is not fully understood. Rapid adenosine release modulates oxygen levels and evoked dopamine release, indicating that adenosine may have a rapid modulatory role. In this review, we outline how FSCV can be used to detect adenosine release, compare FSCV with other techniques used to measure adenosine, and present an overview of adenosine signaling that has been characterized using FSCV. These studies point to a rapid mode of adenosine modulation, whose mechanism and function will continue to be characterized in the future.

  15. Imaging Adenosine Triphosphate (ATP).

    Rajendran, Megha; Dane, Eric; Conley, Jason; Tantama, Mathew


    Adenosine triphosphate (ATP) is a universal mediator of metabolism and signaling across unicellular and multicellular species. There is a fundamental interdependence between the dynamics of ATP and the physiology that occurs inside and outside the cell. Characterizing and understanding ATP dynamics provide valuable mechanistic insight into processes that range from neurotransmission to the chemotaxis of immune cells. Therefore, we require the methodology to interrogate both temporal and spatial components of ATP dynamics from the subcellular to the organismal levels in live specimens. Over the last several decades, a number of molecular probes that are specific to ATP have been developed. These probes have been combined with imaging approaches, particularly optical microscopy, to enable qualitative and quantitative detection of this critical molecule. In this review, we survey current examples of technologies available for visualizing ATP in living cells, and identify areas where new tools and approaches are needed to expand our capabilities.

  16. Protective effect of taurine on hypochlorous acid toxicity to nuclear nucleoside triphosphatase in isolated nuclei from rat liver

    Ju-Xiang Li; Yong-Zheng Pang; Chao-Shu Tang; Zai-Quan Li


    AIM: Taurine has been shown to be an effective scavenger of hypochlorous acid (HOCI). The role of HOCI is well established in tissue damage associated with inflammation and injury. In the present study, the effect of HOCI on nuclear nucleoside triphosphatase of hepatocytes and the ability of taurine to prevent this effect were investigated.METHODS: Isolated hepatic nuclei from rat liver were exposed to HOCI with or without taurine. The NTPase activity on nuclear envelope was assayed using ATP and GTP as substrates, respectively.RESULTS: The first series of experiments evaluated the toxicity of HOCl and the efficacy of taurine to protect NTPase.HOCI at 10-9-5×10-6 mol/L reduced nuclear NTPase activities in a concentration dependent manner (ATP and GTP as substrates) (P<0.01). HOCI at 10-6 mol/L reduced the NTPase activity by 65% (ATP as substrate) and 76% (GTP as substrate). Taurine (10-7 to 10-4 mol/L) was tested for protection against HOCI at 10-6 mol/L and the nuclei treated with 5x10-4 mol/L taurine exhibited only 20% and 12% reduction in NTPase activities compared to untreated controls. A second study was performed comparing taurine to glutathione (GSH). GSH and HOCI at 10-6 mol/L exhibited 46% and 67.4% reduction in NTPase activities compared with control. GSH (10-4 mol/L) which was incubated with the nuclei and HOCi still exhibited 44.2% and 44.8% reduction in NTPase activities of untreated control. Taurine with HOCI only exhibited 15.2% and 17.1% reduction in NTPase activities, which provided more powerful protection against HOCI than GSH. The third experiment was undertaken to evaluate the specificity of taurine against HOCI. Incubation of rat hepatic nuclei with Fe3+/H2O2 (1 m mol/L vS 5μ mol/L) resulted in a decrease in nuclear NTPase activities (P<0.01).When hepatic nuclei were incubated with Tau (10-4 mol/L) and Fe3+/H2O2 (1m mol/L vS 5μ mol/L), nuclear NTPase activities were only slightly increased as compared with that of incubation with Fe3+/H

  17. Supplementation of chitosan alleviates high-fat diet-enhanced lipogenesis in rats via adenosine monophosphate (AMP)-activated protein kinase activation and inhibition of lipogenesis-associated genes.

    Chiu, Chen-Yuan; Chan, Im-Lam; Yang, Tsung-Han; Liu, Shing-Hwa; Chiang, Meng-Tsan


    This study investigated the role of chitosan in lipogenesis in high-fat diet-induced obese rats. The lipogenesis-associated genes and their upstream regulatory proteins were explored. Diet supplementation of chitosan efficiently decreased the increased weights in body, livers, and adipose tissues in high-fat diet-fed rats. Chitosan supplementation significantly raised the lipolysis rate; attenuated the adipocyte hypertrophy, triglyceride accumulation, and lipoprotein lipase activity in epididymal adipose tissues; and decreased hepatic enzyme activities of lipid biosynthesis. Chitosan supplementation significantly activated adenosine monophosphate (AMP)-activated protein kinase (AMPK) phosphorylation and attenuated high-fat diet-induced protein expressions of lipogenic transcription factors (PPAR-γ and SREBP1c) in livers and adipose tissues. Moreover, chitosan supplementation significantly inhibited the expressions of downstream lipogenic genes (FAS, HMGCR, FATP1, and FABP4) in livers and adipose tissues of high-fat diet-fed rats. These results demonstrate for the first time that chitosan supplementation alleviates high-fat diet-enhanced lipogenesis in rats via AMPK activation and lipogenesis-associated gene inhibition.

  18. Feed-Forward Inhibition of CD73 and Upregulation of Adenosine Deaminase Contribute to the Loss of Adenosine Neuromodulation in Postinflammatory Ileitis

    Cátia Vieira


    Full Text Available Purinergic signalling is remarkably plastic during gastrointestinal inflammation. Thus, selective drugs targeting the “purinome” may be helpful for inflammatory gastrointestinal diseases. The myenteric neuromuscular transmission of healthy individuals is fine-tuned and controlled by adenosine acting on A2A excitatory receptors. Here, we investigated the neuromodulatory role of adenosine in TNBS-inflamed longitudinal muscle-myenteric plexus of the rat ileum. Seven-day postinflammation ileitis lacks adenosine neuromodulation, which may contribute to acceleration of gastrointestinal transit. The loss of adenosine neuromodulation results from deficient accumulation of the nucleoside at the myenteric synapse despite the fact that the increases in ATP release were observed. Disparity between ATP outflow and adenosine deficit in postinflammatory ileitis is ascribed to feed-forward inhibition of ecto-5′-nucleotidase/CD73 by high extracellular ATP and/or ADP. Redistribution of NTPDase2, but not of NTPDase3, from ganglion cell bodies to myenteric nerve terminals leads to preferential ADP accumulation from released ATP, thus contributing to the prolonged inhibition of muscle-bound ecto-5′-nucleotidase/CD73 and to the delay of adenosine formation at the inflamed neuromuscular synapse. On the other hand, depression of endogenous adenosine accumulation may also occur due to enhancement of adenosine deaminase activity. Both membrane-bound and soluble forms of ecto-5′-nucleotidase/CD73 and adenosine deaminase were detected in the inflamed myenteric plexus. These findings provide novel therapeutic targets for inflammatory gut motility disorders.

  19. Electroacupuncture improves neuropathic pain Adenosine,adenosine 5'-triphosphate disodium and their receptors perhaps change simultaneously

    Wen Ren; Wenzhan Tu; Songhe Jiang; Ruidong Cheng; Yaping Du


    Applying a stimulating current to acupoints through acupuncture needles-known as electroacupuncture-has the potential to produce analgesic effects in human subjects and experimental animals.When acupuncture was applied in a rat model,adenosine 5'-triphosphate disodium in the extracellular space was broken down into adenosine,which in turn inhibited pain transmission by means of an adenosine A1 receptor-dependent process.Direct injection of an adenosine A1 receptor agonist enhanced the analgesic effect of acupuncture.The analgesic effect of acupuncture appears to be mediated by activation of A1 receptors located on ascending nerves.In neuropathic pain,there is upregulation of P2X purinoceptor 3(P2X3)receptor expression in dorsal root ganglion neurons.Conversely,the onset of mechanical hyperalgesia was diminished and established hyperalgesia was significantly reversed when P2X3 receptor expression was downregulated.The pathways upon which electroacupuncture appear to act are interwoven with pain pathways,and electroacupuncture stimuli converge with impulses originating from painful areas.Electroacupuncture may act via purinergic A1 and P2X3 receptors simultaneously to induce an analgesic effect on neuropathic pain.

  20. The A3 adenosine receptor: history and perspectives.

    Borea, Pier Andrea; Varani, Katia; Vincenzi, Fabrizio; Baraldi, Pier Giovanni; Tabrizi, Mojgan Aghazadeh; Merighi, Stefania; Gessi, Stefania


    By general consensus, the omnipresent purine nucleoside adenosine is considered a major regulator of local tissue function, especially when energy supply fails to meet cellular energy demand. Adenosine mediation involves activation of a family of four G protein-coupled adenosine receptors (ARs): A(1), A(2)A, A(2)B, and A(3). The A(3) adenosine receptor (A(3)AR) is the only adenosine subtype to be overexpressed in inflammatory and cancer cells, thus making it a potential target for therapy. Originally isolated as an orphan receptor, A(3)AR presented a twofold nature under different pathophysiologic conditions: it appeared to be protective/harmful under ischemic conditions, pro/anti-inflammatory, and pro/antitumoral depending on the systems investigated. Until recently, the greatest and most intriguing challenge has been to understand whether, and in which cases, selective A(3) agonists or antagonists would be the best choice. Today, the choice has been made and A(3)AR agonists are now under clinical development for some disorders including rheumatoid arthritis, psoriasis, glaucoma, and hepatocellular carcinoma. More specifically, the interest and relevance of these new agents derives from clinical data demonstrating that A(3)AR agonists are both effective and safe. Thus, it will become apparent in the present review that purine scientists do seem to be getting closer to their goal: the incorporation of adenosine ligands into drugs with the ability to save lives and improve human health.

  1. Pharmacology of the Adenosine A3 Receptor in the Vasculature and Essential Hypertension

    Ho, Ming-Fen; Low, Leanne M.; Rose’Meyer, Roselyn B.


    Background Essential hypertension is considered to be a multifactorial disorder and its aetiology has yet to be clearly identified. As the adenosine receptors have a significant role in mediating vasodilation, alterations in their structures or signalling pathways may be involved in the development of hypertension. This study aimed to measure the expression of adenosine A3 receptors in a range of cardiovascular tissues and determine whether they could be altered with essential hypertension, and to functionally test responses to adenosine A3 receptor agonists in coronary blood vessels using the isolated perfused heart preparation. Methods mRNA samples from cardiovascular tissues and a range of blood vessels were collected from 10 week old male spontaneously hypertensive rats and age-gender matched Wistar rats (n = 8). The Langendorff heart perfusion preparation was used to characterise adenosine A3 receptor mediated coronary vasodilation in the rat heart. Results Adenosine A3 receptor agonists induced coronary vasodilation. The expression of adenosine A3 receptors in cardiovascular tissues was altered in a tissue-specific pattern. Specifically, down-regulation of adenosine A3 receptor expression occurred in hypertensive hearts, which might be associated with attenuated vasodilator responses observed in coronary vessels to adenosine A3 receptor agonists. Conclusions This study demonstrated alterations in the expression of adenosine A3 receptors occurred in a tissue specific mode, and reduced adenosine A3 receptor mediated coronary vasodilation in hearts from spontaneously hypertensive rats. Our findings with regard to changes in the adenosine A3 receptor in hypertensive hearts suggest that adenosine A3 receptor might play a role in the physiopathology of essential hypertension and potentially open the way to pharmacologic manipulation of vasomotor activity by the use of adenosine A3 receptor agonists. PMID:26907173

  2. Contraction induced secretion of VEGF from skeletal muscle cells is mediated by adenosine

    Høier, Birgitte; Olsen, Karina; Nyberg, Michael Permin


    The role of adenosine and contraction for secretion of VEGF in skeletal muscle was investigated in human subjects and rat primary skeletal muscle cells. Microdialysis probes were inserted into the thigh muscle of seven male subjects and dialysate was collected at rest, during infusion of adenosine...... and contraction caused secretion of VEGF (pcontraction induced secretion of VEGF protein was abolished by the A(2B) antagonist enprofyllin and markedly reduced by inhibition of PKA or MAPK. The results demonstrate that adenosine causes secretion of VEGF from human skeletal muscle cells...... and that the contraction induced secretion of VEGF is partially mediated via adenosine acting on A(2B) adenosine receptors. Moreover, the contraction induced secretion of VEGF protein from muscle is dependent on both PKA and MAPK activation, but only the MAPK pathway appears to be adenosine dependent....

  3. [Effects of dopamine and adenosine on regulation of water-electrolyte exchange in Amoeba proteus].

    Bagrov, Ia Iu; Manusova, N B


    Dopamine and adenosine both regulate transport of sodium chloride in the renal tubules in mammals. We have studied the effect of dopamine and adenosine on spontaneous activity of contractile vacuole of Amoeba proteous. Both substances stimulated contractile vacuole. The effect of dopamine was suppressed by D2 receptor antagonist, haloperidol, but not by D1 antagonist, SCH 39166. Adenylate cyclase inhibitor, 2.5-dideoxyadenosine, suppressed the effect of dopamine, but not of adenosine. Inhibitor of protein kinase C, staurosporine, in contrast, blocked the effect of adenosine, but not dopamine. Notably, dopamine opposed effect of adenosine and vice versa. These results suggest that similar effects of dopamine and adenosine could be mediated by different intracellulare mechanisms.

  4. Metformin inhibits nuclear factor-κB activation and inflammatory cytokines expression induced by high glucose via adenosine monophosphate-activated protein kinase activation in rat glomerular mesangial cells in vitro

    Gu Junfei; Ye Shandong; Wang Shan; Sun Wenjia; Hu Yuanyuan


    Background The renoprotective mechanisms of adenosine monophosphate (AMP)-activated protein kinase (AMPK) agonist-metformin have not been stated clearly.We hypothesized that metformin may ameliorate inflammation via AMPK interaction with critical inflammatory cytokines The aim of this study was to observe the effects of metformin on expression of nuclear factor-κB (NF-κB),monocyte chemoattractant protein-1 (MCP-1),intercellular adhesion molecule-1 (ICAM-1) and transforming growth factor-beta 1 (TGF-β1) induced by high glucose (HG) in cultured rat glomerular mesangial cells (MCs).Methods MCs were cultured in the medium with normal concentration glucose (group NG,5.6 mmol/L),high concentration glucose (group HG,25 mmol/L) and different concentrations of metformin (group M1,M2,M3).After 48-hour exposure,the supernatants and MCs were collected.The expression of NF-κB,MCP-1,ICAM-1,and TGF-β1 mRNA was analyzed by real time polymerase chain reaction.Westem blotting was used to detect the expression of AMPK,phospho-Thr-172 AMPK (p-AMPK),NF-κB p65,MCP-1,ICAM-1,and TGF-β1 protein.Results After stimulated by HG,the expression of NF-κB,MCP-1,ICAM-1,TGF-β1 mRNA and protein of MCs in group HG increased significantly compared with group NG (P <0.05).Both genes and protein expression of NF-κB,MCP-1,ICAM-1,TGF-β1 of MCs induced by high glucose were markedly reduced after metformin treatment in a dose-dependent manner (P <0.05).The expression of p-AMPK increased with the rising of metformin concentration,presenting the opposite trend,while the level of total-AMPK protein was unchanged with exposure to HG or metformin.Conlusion Metformin can suppress the expression of NF-κB,MCP-1,ICAM-1 and TGF-β1 of glomerular MCs induced by high glucose via AMPK activation,which may partlv contribute to its reno-protection.

  5. Berberine treatment prevents cardiac dysfunction and remodeling through activation of 5'-adenosine monophosphate-activated protein kinase in type 2 diabetic rats and in palmitate-induced hypertrophic H9c2 cells.

    Chang, Wenguang; Zhang, Ming; Meng, Zhaojie; Yu, Yang; Yao, Fan; Hatch, Grant M; Chen, Li


    Diabetic cardiomyopathy is the major cause of death in type 2 diabetic patients. Berberine is an isoquinoline alkaloid extract from traditional chinese herbs and its hypoglycemic and hypolipidemic effects make it a promising drug for treatment of type 2 diabetes. We examined if berberine improved cardiac function and attenuated cardiac hypertrophy and fibrosis in high fat diet and streptozotocin induced-type 2 diabetic rats in vivo and reduced expression of hypertrophy markers in palmitate-induced hypertrophic H9c2 cells in vitro. Treatment of diabetic animals with berberine partially improved cardiac function and restored fasting blood insulin, fasting blood glucose, total cholesterol, and triglyceride levels to that of control. In addition, berberine treatment of diabetic animals increased cardiac 5'-adenosine monophosphate-activated protein kinase (AMPK) and protein kinase B (AKT) activation and reduced glycogen synthase kinase 3 beta (GSK3β) activation compared to control. Palmitate incubation of H9c2 cells resulted in cellular hypertrophy and decreased expression of alpha-myosin heavy chain (α-MHC) and increased expression of beta-myosin heavy chain (β-MHC) compared to controls. Berberine treatment of palmitate-incubated H9c2 cells reduced hypertrophy, increased α-MHC expression and decreased β-MHC expression. In addition, berberine treatment of palmitate-incubated H9c2 cells increased AMPK and AKT activation and reduced GSK3β activation. The presence of the AMPK inhibitor Compound C attenuated the effects of berberine. The results strongly indicate that berberine treatment may be protective against the development of diabetic cardiomyopathy.

  6. Some selective serotonin reuptake inhibitors inhibit dynamin I guanosine triphosphatase (GTPase).

    Otomo, Masahiro; Takahashi, Kiyofumi; Miyoshi, Hiroshi; Osada, Kenichi; Nakashima, Hideki; Yamaguchi, Noboru


    Neuronal dynamin I plays a critical role in the recycling of synaptic vesicles, and thus in nervous system function. We expressed and purified dynamin I to explore potentially clinically useful endocytosis inhibitors and to examine the mechanism of their action. We estimated the IC(50) of nineteen psychotropic drugs for dynamin I. The IC(50) values of two selective serotonin reuptake inhibitors (sertraline and fluvoxamine) were 7.3+/-1.0 and 14.7+/-1.6 microM, respectively. Kinetic analyses revealed that fluvoxamine is a noncompetitive inhibitor of dynamin I guanosine triphosphatase (GTPase) with respect to guanosine 5'-triphosphate (GTP) and a competitive inhibitor with respect to L-phosphatidylserine (PS). Fluvoxamine may compete with PS for binding to the pleckstrin homology domain of dynamin I. On the other hand, sertraline was a mixed type inhibitor with respect to both GTP and PS. Our results indicate that sertraline and fluvoxamine may regulate the transportation of neurotransmitters by modulating synaptic vesicle endocytosis via the inhibition of dynamin I GTPase.

  7. Diversification of the RAB Guanosine Triphosphatase Family in Dicots and Monocots


    RAB guanosine triphosphatases (GTPases) are key regulators of vesicle trafficking and are essential to the growth and development of all eukaryotic cells. During evolution, the RAB family has expanded in different patterns to facilitate distinct cellular, developmental and physiological adaptations. Yeast has only 11 family members, whereas mammalian RABs have expanded to 18 RAB subfamilies. Plant RABs have diversified primarily by duplicating members within a single subfamily. Plant RABs are divided into eight subfamilies, corresponding to mammalian RAB1, RAB2, RAB5, RAB6,RAB7, RAB8, RAB11 and RAB18. Functional diversification of these is exemplified by the RAB11s, orthologs of which are partitioned into unique cell compartments in plants where they function to transport vesicles during localized tip growth.Similarly, the RAB2 family in grasses is likely involved in vesicle secretion associated with wall expansion, as determined by analysis of over-expression mutants. We propose that dicots and monocots have also diverged in their RAB profiles to accommodate unique cellular functions between the two groups. Here we present a bioinformatics analysis comparing the RAB sub-families of rice, maize and Arabidopsis. These results will guide future functional studies to test for the role of diversification of subfamilies unique to monocots compared to dicots.

  8. Role of A3 adenosine receptor in diabetic neuropathy.

    Yan, Heng; Zhang, Enshui; Feng, Chang; Zhao, Xin


    Neuropathy is the most common diabetic complication. Although the A1 and A2A adenosine receptors are important pharmacological targets in alleviating diabetic neuropathy, the role of the A3 adenosine receptor remains unknown. Because the A3 adenosine receptor regulates pain induced by chronic constriction injury or chemotherapy, its stimulation might also attenuate diabetic neuropathy. This study examines the effects of systemic treatment with the A3 adenosine receptor agonist 1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-β-d-ribofuranuronamide (IB-MECA) on diabetic neuropathy and explores the putative mechanisms underlying its pharmacological effects. We show that IB-MECA alleviated mechanical hyperalgesia and thermal hypoalgesia in mice 2 weeks but not 4 weeks after streptozocin (STZ) treatment. Furthermore, IB-MECA prevented the reduction in sciatic motor nerve conduction velocity and sensory nerve conduction velocity in diabetic mice 2 weeks but not 4 weeks after STZ treatment. Similarly, IB-MECA inhibited the activation of nuclear factor-κB and decreased the generation of tumor necrosis factor-α in the spinal cord of mice 2 weeks but not 4 weeks after STZ treatment. These phenomena were associated with reduction of A3 adenosine receptor expression in the spinal cord after long-term diabetes. Our results suggest that the A3 adenosine receptor plays a critical role in regulating diabetic neuropathy and that reduction in A3 adenosine receptor expression/function might contribute to the progression of diabetic neuropathy. © 2016 Wiley Periodicals, Inc.

  9. Correlation between blood adenosine metabolism and sleep in humans.

    Díaz-Muñoz, M; Hernández-Muñoz, R; Suárez, J; Vidrio, S; Yááñez, L; Aguilar-Roblero, R; Rosenthal, L; Villalobos, L; Fernández-Cancino, F; Drucker-Colín, R; Chagoya De Sanchez, V


    Blood adenosine metabolism, including metabolites and metabolizing enzymes, was studied during the sleep period in human volunteers. Searching for significant correlations among biochemical parameters found: adenosine with state 1 of slow-wave sleep (SWS); activity of 5'-nucleotidase with state 2 of SWS; inosine and AMP with state 3-4 of SWS; and activity of 5'-nucleotidase and lactate with REM sleep. The correlations were detected in all of the subjects that presented normal hypnograms, but not in those who had fragmented sleep the night of the experiment. The data demonstrate that it is possible to obtain information of complex brain operations such as sleep by measuring biochemical parameters in blood. The results strengthen the notion of a role played by adenosine, its metabolites and metabolizing enzymes, during each of the stages that constitute the sleep process in humans.

  10. Activation of the adenosine A2A receptor exacerbates experimental autoimmune neuritis in Lewis rats in association with enhanced humoral immunity.

    Zhang, Min; Li, Xiao-Li; Li, Heng; Wang, Shan; Wang, Cong-Cong; Yue, Long-Tao; Xu, Hua; Zhang, Peng; Chen, Hui; Yang, Bing; Duan, Rui-Sheng


    Accumulated evidence demonstrated that Adenosine A2A receptor (A2AR) is involved in the inflammatory diseases. In the present study, we showed that a selective A2AR agonist, CGS21680, exacerbated experimental autoimmune neuritis in Lewis rats induced with bovine peripheral myelin. The exacerbation was accompanied with reduced CD4(+)Foxp3(+) T cells, increased CD4(+)CXCR5(+) T cells, B cells, dendritic cells and antigen-specific autoantibodies, which is possibly due to the inhibition of IL-2 induced by CGS21680. Combined with previous studies, our data indicate that the effects of A2AR stimulation in vivo are variable in different diseases. Caution should be taken in the use of A2AR agonists.

  11. Adenosine A(1) Receptors in the Central Nervous System : Their Functions in Health and Disease, and Possible Elucidation by PET Imaging

    Paul, S.; Elsinga, P. H.; Ishiwata, K.; Dierckx, R. A. J. O.; van Waarde, A.


    Adenosine is a neuromodulator with several functions in the central nervous system (CNS), such as inhibition of neuronal activity in many signaling pathways. Most of the sedating, anxiolytic, seizure-inhibiting and protective actions of adenosine are mediated by adenosine A(1) receptors (A(1)R) on t

  12. The role of muscarinic receptors in the beneficial effects of adenosine against myocardial reperfusion injury in rats.

    Lei Sun

    Full Text Available Adenosine, a catabolite of ATP, displays a wide variety of effects in the heart including regulation of cardiac response to myocardial ischemia and reperfusion injury. Nonetheless, the precise mechanism of adenosine-induced cardioprotection is still elusive. Isolated Sprague-Dawley rat hearts underwent 30 min global ischemia and 120 min reperfusion using a Langendorff apparatus. Both adenosine and acetylcholine treatment recovered the post-reperfusion cardiac function associated with adenosine and muscarinic receptors activation. Simultaneous administration of adenosine and acetylcholine failed to exert any additive protective effect, suggesting a shared mechanism between the two. Our data further revealed a cross-talk between the adenosine and acetylcholine receptor signaling in reperfused rat hearts. Interestingly, the selective M(2 muscarinic acetylcholine receptor antagonist methoctramine significantly attenuated the cardioprotective effect of adenosine. In addition, treatment with adenosine upregulated the expression and the maximal binding capacity of muscarinic acetylcholine receptor, which were inhibited by the selective A(1 adenosine receptor antagonist 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX and the nitric oxide synthase inhibitor N(ω-nitro-L-arginine methyl ester (L-NAME. These data suggested a possible functional coupling between the adenosine and muscarinic receptors behind the observed cardioprotection. Furthermore, nitric oxide was found involved in triggering the response to each of the two receptor agonist. In summary, there may be a cross-talk between the adenosine and muscarinic receptors in ischemic/reperfused myocardium with nitric oxide synthase might serve as the distal converging point. In addition, adenosine contributes to the invigorating effect of adenosine on muscarinic receptor thereby prompting to regulation of cardiac function. These findings argue for a potentially novel mechanism behind the adenosine

  13. Regulation of adenosine levels during cerebral ischemia

    Stephanie CHU; Wei XIONG; Dali ZHANG; Hanifi SOYLU; Chao SUN; Benedict C ALBENSI; Fiona E PARKINSON


    Adenosine is a neuromodulator with its level increasing up to 100-fold during ischemic events,and attenuates the excitotoxic neuronal injury.Adenosine is produced both intracellularly and extracellularly,and nucleoside transport proteins transfer adenosine across plasma membranes.Adenosine levels and receptor-mediated effects of adenosine are regulated by intracellular ATP consumption,cellular release of ATP,metabolism of extracellular ATP (and other adenine nucleotides),adenosine influx,adenosine efflux and adenosine metabolism.Recent studies have used genetically modified mice to investigate the relative contributions of intra-and extracellular pathways for adenosine formation.The importance of cortical or hippocampal neurons as a source or a sink of adenosine under basal and hypoxic/ischemic conditions was addressed through the use of transgenic mice expressing human equilibrative nucleoside transporter 1 (hENT1) under the control of a promoter for neuron-specific enolase.From these studies,we conclude that ATP consumption within neurons is the primary source of adenosine in neuronal cultures,but not in hippocampal slices or in vivo mice exposed to ischemic conditions.

  14. Evidence for an A2/Ra adenosine receptor in the guinea-pig trachea

    Brown, C.M.; Collis, M.G.


    1 An attempt was made to determine whether the extracellular adenosine receptor that mediates relaxation in the guinea-pig trachea is of the A1/Ri or A2/Ra subtype. 2 Dose-response curves to adenosine and a number of 5′- and N6-substituted analogues were constructed for the isolated guinea-pig trachea, contracted with carbachol. 3 The 5′-substituted analogues of adenosine were the most potent compounds tested, the order of potency being 5′-N-cyclopropylcarboxamide adenosine (NCPCA) > 5′-N-ethylcarboxamide adenosine (NECA) > 2-chloroadenosine > L-N6-phenylisopropyladenosine (L-PIA) > adenosine > D-N6-phenylisopropyladenosine (D-PIA). 4 The difference in potency between the stereoisomers D- and L-PIA on the isolated trachea was at the most five fold. 5 Responses to low doses of adenosine and its analogues were attenuated after treatment with either theophylline or 8-phenyltheophylline. The responses to 2-chloroadenosine were affected to a lesser extent than were those to the other purines. 6 Adenosine transport inhibitors, dipyridamole and dilazep, potentiated responses to adenosine, did not affect those to NCPCA, NECA, L-PIA and D-PIA but significantly reduced the responses to high doses of 2-chloroadenosine. 7 Relaxations evoked by 9-β-D-xylofuranosyladenosine which can activate intracellular but not extracellular adenosine receptors, were attenuated by dipyridamole but unaffected by 8-phenyltheophylline. 8 The results support the existence of an extracellular A2/Ra subtype of adenosine receptor and an intracellular purine-sensitive site, both of which mediate relaxation. PMID:6286021

  15. Characterization of spontaneous, transient adenosine release in the caudate-putamen and prefrontal cortex.

    Nguyen, Michael D; Lee, Scott T; Ross, Ashley E; Ryals, Matthew; Choudhry, Vishesh I; Venton, B Jill


    Adenosine is a neuroprotective agent that inhibits neuronal activity and modulates neurotransmission. Previous research has shown adenosine gradually accumulates during pathologies such as stroke and regulates neurotransmission on the minute-to-hour time scale. Our lab developed a method using carbon-fiber microelectrodes to directly measure adenosine changes on a sub-second time scale with fast-scan cyclic voltammetry (FSCV). Recently, adenosine release lasting a couple of seconds has been found in murine spinal cord slices. In this study, we characterized spontaneous, transient adenosine release in vivo, in the caudate-putamen and prefrontal cortex of anesthetized rats. The average concentration of adenosine release was 0.17±0.01 µM in the caudate and 0.19±0.01 µM in the prefrontal cortex, although the range was large, from 0.04 to 3.2 µM. The average duration of spontaneous adenosine release was 2.9±0.1 seconds and 2.8±0.1 seconds in the caudate and prefrontal cortex, respectively. The concentration and number of transients detected do not change over a four hour period, suggesting spontaneous events are not caused by electrode implantation. The frequency of adenosine transients was higher in the prefrontal cortex than the caudate-putamen and was modulated by A1 receptors. The A1 antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine, 6 mg/kg i.p.) increased the frequency of spontaneous adenosine release, while the A1 agonist CPA (N(6)-cyclopentyladenosine, 1 mg/kg i.p.) decreased the frequency. These findings are a paradigm shift for understanding the time course of adenosine signaling, demonstrating that there is a rapid mode of adenosine signaling that could cause transient, local neuromodulation.

  16. Adenosine Inhibits the Excitatory Synaptic Inputs to Basal Forebrain Cholinergic, GABAergic and Parvalbumin Neurons in mice

    Chun eYang


    Full Text Available Coffee and tea contain the stimulants caffeine and theophylline. These compounds act as antagonists of adenosine receptors. Adenosine promotes sleep and its extracellular concentration rises in association with prolonged wakefulness, particularly in the basal forebrain (BF region involved in activating the cerebral cortex. However, the effect of adenosine on identified BF neurons, especially non-cholinergic neurons, is incompletely understood. Here we used whole-cell patch-clamp recordings in mouse brain slices prepared from two validated transgenic mouse lines with fluorescent proteins expressed in GABAergic or parvalbumin (PV neurons to determine the effect of adenosine. Whole-cell recordings were made BF cholinergic neurons and from BF GABAergic & PV neurons with the size (>20 µm and intrinsic membrane properties (prominent H-currents corresponding to cortically projecting neurons. A brief (2 min bath application of adenosine (100 μM decreased the frequency but not the amplitude of spontaneous excitatory postsynaptic currents in all groups of BF cholinergic, GABAergic and PV neurons we recorded. In addition, adenosine decreased the frequency of miniature EPSCs in BF cholinergic neurons. Adenosine had no effect on the frequency of spontaneous inhibitory postsynaptic currents in cholinergic neurons or GABAergic neurons with large H-currents but reduced them in a group of GABAergic neurons with smaller H-currents. All effects of adenosine were blocked by a selective, adenosine A1 receptor antagonist, cyclopentyltheophylline (CPT, 1 μM. Adenosine had no postsynaptic effects. Taken together, our work suggests that adenosine promotes sleep by an A1-receptor mediated inhibition of glutamatergic inputs to cortically-projecting cholinergic and GABA/PV neurons. Conversely, caffeine and theophylline promote attentive wakefulness by inhibiting these A1 receptors in BF thereby promoting the high-frequency oscillations in the cortex required for

  17. Small-Animal PET Study of Adenosine A(1) Receptors in Rat Brain : Blocking Receptors and Raising Extracellular Adenosine

    Paul, Soumen; Khanapur, Shivashankar; Rybczynska, Anna A.; Kwizera, Chantal; Sijbesma, Jurgen W. A.; Ishiwata, Kiichi; Willemsen, Antoon T. M.; Elsinga, Philip H.; Dierckx, Rudi A. J. O.; van Waarde, Aren


    Activation of adenosine A(1) receptors (A(1)R) in the brain causes sedation, reduces anxiety, inhibits seizures, and promotes neuroprotection. Cerebral A(1)R can be visualized using 8-dicyclopropylmethyl-1-C-11-methyl-3-propyl-xanthine (C-11-MPDX) and PET. This study aims to test whether C-11-MPDX c

  18. Ras activation by SOS

    Iversen, Lars; Tu, Hsiung-Lin; Lin, Wan-Chen;


    Activation of the small guanosine triphosphatase H-Ras by the exchange factor Son of Sevenless (SOS) is an important hub for signal transduction. Multiple layers of regulation, through protein and membrane interactions, govern activity of SOS. We characterized the specific activity of individual ...

  19. Exercise-induced increase in interstitial bradykinin and adenosine concentrations in skeletal muscle and peritendinous tissue in humans

    Langberg, H; Bjørn, C; Boushel, Robert Christopher


    increased both in muscle (from 0.48 +/- 0.07 micromol l(-1) to 1.59 +/- 0.35 micromol l(-1); P muscular activity increases the interstitial concentrations...... of bradykinin and adenosine in both skeletal muscle and the connective tissue around its adjacent tendon. These findings support a role for bradykinin and adenosine in exercise-induced hyperaemia in skeletal muscle and suggest that bradykinin and adenosine are potential regulators of blood flow in peritendinous...

  20. Pathologic overproduction: the bad side of adenosine.

    Borea, Pier Andrea; Gessi, Stefania; Merighi, Stefania; Vincenzi, Fabrizio; Varani, Katia


    Adenosine is an endogenous ubiquitous purine nucleoside, increased by hypoxia, ischemia and tissue damage that mediates a number of physiopathological effects by interacting with four G-protein-coupled receptors, identified as A1 , A2A , A2B , and A3 . Physiological and acutely-increased adenosine is associated with beneficial effects mostly including vasodilation and decrease of inflammation. In contrast chronic overproduction of adenosine occurs in important pathological states, where long lasting increases in the nucleoside levels are responsible for the bad side of adenosine associated with chronic inflammation, fibrosis and organ damage. In this review we describe and critically discuss the pathologic overproduction of adenosine analysing when, where and how adenosine exerts its detrimental effects through the body.

  1. CD39/adenosine pathway is involved in AIDS progression.

    Maria Nikolova


    Full Text Available HIV-1 infection is characterized by a chronic activation of the immune system and suppressed function of T lymphocytes. Regulatory CD4+ CD25(high FoxP3+CD127(low T cells (Treg play a key role in both conditions. Here, we show that HIV-1 positive patients have a significant increase of Treg-associated expression of CD39/ENTPD1, an ectoenzyme which in concert with CD73 generates adenosine. We show in vitro that the CD39/adenosine axis is involved in Treg suppression in HIV infection. Treg inhibitory effects are relieved by CD39 down modulation and are reproduced by an adenosine-agonist in accordance with a higher expression of the adenosine A2A receptor on patients' T cells. Notably, the expansion of the Treg CD39+ correlates with the level of immune activation and lower CD4+ counts in HIV-1 infected patients. Finally, in a genetic association study performed in three different cohorts, we identified a CD39 gene polymorphism that was associated with down-modulated CD39 expression and a slower progression to AIDS.

  2. Inhibition by adenosine of histamine and leukotriene release from human basophils.

    Peachell, P T; Lichtenstein, L M; Schleimer, R P


    Adenosine inhibited the release of histamine and leukotriene C4 (LTC4) from immunologically-activated basophils in a dose-dependent manner. Structural congeners of adenosine also attenuated the elaboration of these two mediators from stimulated basophils and a rank order of potency for the inhibition was observed following the sequence 2-chloroadenosine greater than or equal to N-ethylcarboxamidoadenosine (NECA) greater than adenosine greater than or equal to R-phenylisopropyladenosine (R-PIA) greater than or equal to S-PIA. These same nucleosides modulated the generation of LTC4 more potently than the release of histamine. A number of methylxanthines, which are antagonists of cell surface adenosine receptors, reversed the inhibition by adenosine and its congeners of the release of both histamine and LTC4 to varying extents. Dipyridamole and nitrobenzylthioinosine (NBTI), agents that block the intracellular uptake of adenosine, antagonized the inhibition of histamine release by adenosine (and 2-chloroadenosine) but failed to reverse the attenuation of LTC4 generation by the nucleoside. These same uptake blockers were unable to antagonize the inhibitory effects of NECA on either histamine or LTC4 release. In purified basophils, NECA and R-PIA, and in that order of decreasing reactivity, increased total cell cyclic adenosine monophosphate (cAMP) levels and inhibited the stimulated release of mediators. In total, these results suggest that the basophil possesses a cell surface adenosine receptor which, on the basis of both pharmacological and biochemical criteria, most closely conforms to an A2/Ra-like receptor. However, in addition to an interaction at the cell surface, studies with agents that block the intracellular uptake of adenosine suggest that the nucleoside may also exert intracellular effects when countering the release of histamine (but not LTC4).

  3. Targeting the inflammasome and adenosine type-3 receptors improves outcome of antibiotic therapy in murine anthrax

    Popov, Serguei G.; Popova, Taissia G.; Kashanchi, Fatah; Bailey, Charles


    AIM: To establish whether activation of adenosine type-3 receptors (A3Rs) and inhibition of interleukin-1β-induced inflammation is beneficial in combination with antibiotic therapy to increase survival of mice challenged with anthrax spores.

  4. Effect of insulin and glucose on adenosine metabolizing enzymes in human B lymphocytes.

    Kocbuch, Katarzyna; Sakowicz-Burkiewicz, Monika; Grden, Marzena; Szutowicz, Andrzej; Pawelczyk, Tadeusz


    In diabetes several aspects of immunity are altered, including the immunomodulatory action of adenosine. Our study was undertaken to investigate the effect of different glucose and insulin concentrations on activities of adenosine metabolizing enzymes in human B lymphocytes line SKW 6.4. The activity of adenosine deaminase in the cytosolic fraction was very low and was not affected by different glucose concentration, but in the membrane fraction of cells cultured with 25 mM glucose it was decreased by about 35% comparing to the activity in cells maintained in 5 mM glucose, irrespective of insulin concentration. The activities of 5'-nucleotidase (5'-NT) and ecto-5'-NT in SKW 6.4 cells depended on insulin concentration, but not on glucose. Cells cultured with 10(-8) M insulin displayed an about 60% lower activity of cytosolic 5'-NT comparing to cells maintained at 10(-11) M insulin. The activity of ecto-5'-NT was decreased by about 70% in cells cultured with 10(-8) M insulin comparing to cells grown in 10(-11) M insulin. Neither insulin nor glucose had an effect on adenosine kinase (AK) activity in SKW 6.4 cells or in human B cells isolated from peripheral blood. The extracellular level of adenosine and inosine during accelerated catabolism of cellular ATP depended on glucose, but not on insulin concentration. Concluding, our study demonstrates that glucose and insulin differentially affect the activities of adenosine metabolizing enzymes in human B lymphocytes, but changes in those activities do not correlate with the adenosine level in cell media during accelerated ATP catabolism, implying that nucleoside transport is the primary factor determining the extracellular level of adenosine.

  5. Discovery and Preclinical Characterization of 6-Chloro-5-[4-(1-hydroxycyclobutyl)phenyl]-1H-indole-3-carboxylic Acid (PF-06409577), a Direct Activator of Adenosine Monophosphate-activated Protein Kinase (AMPK), for the Potential Treatment of Diabetic Nephropathy.

    Cameron, Kimberly O; Kung, Daniel W; Kalgutkar, Amit S; Kurumbail, Ravi G; Miller, Russell; Salatto, Christopher T; Ward, Jessica; Withka, Jane M; Bhattacharya, Samit K; Boehm, Markus; Borzilleri, Kris A; Brown, Janice A; Calabrese, Matthew; Caspers, Nicole L; Cokorinos, Emily; Conn, Edward L; Dowling, Matthew S; Edmonds, David J; Eng, Heather; Fernando, Dilinie P; Frisbie, Richard; Hepworth, David; Landro, James; Mao, Yuxia; Rajamohan, Francis; Reyes, Allan R; Rose, Colin R; Ryder, Tim; Shavnya, Andre; Smith, Aaron C; Tu, Meihua; Wolford, Angela C; Xiao, Jun


    Adenosine monophosphate-activated protein kinase (AMPK) is a protein kinase involved in maintaining energy homeostasis within cells. On the basis of human genetic association data, AMPK activators were pursued for the treatment of diabetic nephropathy. Identification of an indazole amide high throughput screening (HTS) hit followed by truncation to its minimal pharmacophore provided an indazole acid lead compound. Optimization of the core and aryl appendage improved oral absorption and culminated in the identification of indole acid, PF-06409577 (7). Compound 7 was advanced to first-in-human trials for the treatment of diabetic nephropathy.

  6. Severe hemorrhage attenuates cardiopulmonary chemoreflex control of regional sympathetic outputs via NTS adenosine receptors.

    Minic, Zeljka; Li, Cailian; O'Leary, Donal S; Scislo, Tadeusz J


    Selective stimulation of inhibitory A1 and facilitatory A2a adenosine receptor subtypes located in the nucleus of the solitary tract (NTS) powerfully inhibits cardiopulmonary chemoreflex (CCR) control of regional sympathetic outputs via different mechanisms: direct inhibition of glutamate release and facilitation of an inhibitory neurotransmitter release, respectively. However, it remains unknown whether adenosine naturally released into the NTS has similar inhibitory effects on the CCR as the exogenous agonists do. Our previous study showed that adenosine is released into the NTS during severe hemorrhage and contributes to reciprocal changes of renal (decreases) and adrenal (increases) sympathetic nerve activity observed in this setting. Both A1 and A2a adenosine receptors are involved. Therefore, we tested the hypothesis that, during severe hemorrhage, CCR control of the two sympathetic outputs is attenuated by adenosine naturally released into the NTS. We compared renal and adrenal sympathoinhibitory responses evoked by right atrial injections of 5HT3 receptor agonist phenylbiguanide (2-8 μg/kg) under control conditions, during hemorrhage, and during hemorrhage preceded by blockade of NTS adenosine receptors with bilateral microinjections of 8-(p-sulfophenyl) theophylline (1 nmol/100 nl) in urethane/chloralose anesthetized rats. CCR-mediated inhibition of renal and adrenal sympathetic activity was significantly attenuated during severe hemorrhage despite reciprocal changes in the baseline activity levels, and this attenuation was removed by bilateral blockade of adenosine receptors in the caudal NTS. This confirmed that adenosine endogenously released into the NTS has a similar modulatory effect on integration of cardiovascular reflexes as stimulation of NTS adenosine receptors with exogenous agonists.

  7. Adenosine, Energy Metabolism, and Sleep

    Tarja Porkka-Heiskanen


    Full Text Available While the exact function of sleep remains unknown, it is evident that sleep was developed early in phylogenesis and represents an ancient and vital strategy for survival. Several pieces of evidence suggest that the function of sleep is associated with energy metabolism, saving of energy, and replenishment of energy stores. Prolonged wakefulness induces signs of energy depletion in the brain, while experimentally induced, local energy depletion induces increase in sleep, similarly as would a period of prolonged wakefulness. The key molecule in the induction of sleep appears to be adenosine, which induces sleep locally in the basal forebrain.

  8. Role of adenosine A2b receptor overexpression in tumor progression.

    Sepúlveda, Cesar; Palomo, Iván; Fuentes, Eduardo


    The adenosine A2b receptor is a G-protein coupled receptor. Its activation occurs with high extracellular adenosine concentration, for example in inflammation or hypoxia. These conditions are generated in the tumor environment. Studies show that A2b receptor is overexpressed in various tumor lines and biopsies from patients with different cancers. This suggests that A2b receptor can be used by tumor cells to promote progression. Thus A2b participates in different events, such as angiogenesis and metastasis, besides exerting immunomodulatory effects that protect tumor cells. Therefore, adenosine A2b receptor appears as an interesting therapeutic target for cancer treatment.

  9. An STS in the human adenosine deaminase gene (located 20q12-q13. 11)

    Freeman, B.C.; States, J.C. (Wayne State Univ., Detroit, MI (United States))


    The human adenosine deaminase gene has been characterized in detail. The adenosine gene product, as part of the purine catabolic pathway, catalyzes the irreversible deamination of adenosine and deoxyadenosine. Deficiency of this activity in humans is associated with an autosomal recessive form of severe combined immunodeficiency disease. Recently, this genetic deficiency disease has been targeted for the first attempts at gene therapy in humans. Using the polymerase chain reaction (PCR), a fragment of the expected size (160 bp) was amplified from human genomic DNA.

  10. Vasodilator effects of adenosine on retinal arterioles in streptozotocin-induced diabetic rats.

    Nakazawa, Taisuke; Mori, Asami; Saito, Maki; Sakamoto, Kenji; Nakahara, Tsutomu; Ishii, Kunio


    Adenosine is a potent vasodilator of retinal blood vessels and is implicated to be a major regulator of retinal blood flow during metabolic stress, but little is known about the impact of diabetes on the role of adenosine in regulation of retinal hemodynamics. Therefore, we examined how diabetes affects adenosine-induced vasodilation of retinal arterioles. Male Wistar rats were treated with streptozotocin (80 mg/kg, intraperitoneally), and experiments were performed 6-8 weeks later. Rats were treated with tetrodotoxin (50 microg/kg, intravenously [i.v.]) to eliminate any nerve activity and prevent movement of the eye and infused with methoxamine continuously to maintain adequate systemic circulation. Fundus images were captured with a digital camera that was equipped with a special objective lens, and diameters of retinal arterioles were measured. Adenosine increased diameters of retinal arterioles and decreased systemic blood pressure. These responses were significantly attenuated by the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (30 mg/kg, i.v.) and the adenosine triphosphate-dependent K+ (K(ATP)) channel blocker glibenclamide (20 mg/kg, i.v.). The depressor responses to adenosine were reduced in diabetic rats, whereas diabetes did not alter vasodilation of retinal arterioles to adenosine. In contrast, both depressor response and vasodilation of retinal arteriole to acetylcholine were reduced in diabetic rats. The retinal vasodilator responses to adenosine and acetylcholine observed in diabetic rats were diminished by N(G)-nitro-L-arginine methyl ester. There were no differences in the responses to pinacidil, a K(ATP) channel opener, between the diabetic and nondiabetic rats. These results suggest that both the activation of nitric oxide synthase and opening of K(ATP) channels contribute to the vasodilator effects of adenosine in rats in vivo. However, diabetes has no significant impact on the vasodilation mediated by these mechanisms in

  11. Antidiabetic sulfonylureas and cAMP cooperatively activate Epac2A.

    Takahashi, Toshimasa; Shibasaki, Tadao; Takahashi, Harumi; Sugawara, Kenji; Ono, Aika; Inoue, Naoko; Furuya, Toshio; Seino, Susumu


    Sulfonylureas are widely used drugs for treating insulin deficiency in patients with type 2 diabetes. Sulfonylureas bind to the regulatory subunit of the pancreatic β cell potassium channel that controls insulin secretion. Sulfonylureas also bind to and activate Epac2A, a member of the Epac family of cyclic adenosine monophosphate (cAMP)-binding proteins that promote insulin secretion through activation of the Ras-like guanosine triphosphatase Rap1. Using molecular docking simulation, we identified amino acid residues in one of two cyclic nucleotide-binding domains, cNBD-A, in Epac2A predicted to mediate the interaction with sulfonylureas. We confirmed the importance of the identified residues by site-directed mutagenesis and analysis of the response of the mutants to sulfonylureas using two assays: changes in fluorescence resonance energy transfer (FRET) of an Epac2A-FRET biosensor and direct sulfonylurea-binding experiments. These residues were also required for the sulfonylurea-dependent Rap1 activation by Epac2A. Binding of sulfonylureas to Epac2A depended on the concentration of cAMP and the structures of the drugs. Sulfonylureas and cAMP cooperatively activated Epac2A through binding to cNBD-A and cNBD-B, respectively. Our data suggest that sulfonylureas stabilize Epac2A in its open, active state and provide insight for the development of drugs that target Epac2A.

  12. Inosine triphosphatase allele frequency and association with ribavirin-induced anaemia in Brazilian patients receiving antiviral therapy for chronic hepatitis C

    Nathália Delvaux


    Full Text Available Inosine triphosphatase (ITPA single nucleotide polymorphisms (SNPs are strongly associated with protection against ribavirin (RBV-induced anaemia in European, American and Asian patients; however, there is a paucity of data for Brazilian patients. The aim of this study was to evaluate the ITPA SNP (rs7270101/rs1127354 frequency in healthy and hepatitis C virus (HCV-infected patients from Brazil and the association with the development of severe anaemia during antiviral therapy. ITPA SNPs were determined in 200 HCV infected patients and 100 healthy individuals by sequencing. Biochemical parameters and haemoglobin (Hb levels were analysed in 97 patients who underwent antiviral therapy. A combination of AArs7270101+CCrs1127354 (100% ITPase activity was observed in 236/300 individuals. Anaemia was observed in 87.5% and 86.2% of treated patients with AA (rs7270101 and CC genotypes (rs1127354, respectively. Men with AA (rs7270101 showed a considerable reduction in Hb at week 12 compared to those with AC/CC (p = 0.1475. In women, there was no influence of genotype (p = 0.5295. For rs1127354, men with the CC genotype also showed a sudden reduction in Hb compared to those with AC. Allelic distribution of rs7270101 and rs1127354 shows high rates of the genotypes AA and CC, respectively, suggesting that the study population had a great propensity for developing RBV-induced anaemia. A progressive Hb reduction during treatment was observed; however, this reduction was greater in men at week 12 than in women.

  13. Inosine triphosphatase allele frequency and association with ribavirin-induced anaemia in Brazilian patients receiving antiviral therapy for chronic hepatitis C

    Delvaux, Nathália; da Costa, Vanessa Duarte; da Costa, Maristella Matos; Villar, Livia Melo; Coelho, Henrique Sérgio Moraes; Esberard, Eliane Bordalo Cathalá; Flores, Priscila Pollo; Brandão-Mello, Carlos Eduardo; Villela-Nogueira, Cristiane Alves; de Almeida, Adilson José; Lampe, Elisabeth


    Inosine triphosphatase (ITPA) single nucleotide polymorphisms (SNPs) are strongly associated with protection against ribavirin (RBV)-induced anaemia in European, American and Asian patients; however, there is a paucity of data for Brazilian patients. The aim of this study was to evaluate the ITPA SNP (rs7270101/rs1127354) frequency in healthy and hepatitis C virus (HCV)-infected patients from Brazil and the association with the development of severe anaemia during antiviral therapy. ITPA SNPs were determined in 200 HCV infected patients and 100 healthy individuals by sequencing. Biochemical parameters and haemoglobin (Hb) levels were analysed in 97 patients who underwent antiviral therapy. A combination of AArs7270101+CCrs1127354 (100% ITPase activity) was observed in 236/300 individuals. Anaemia was observed in 87.5% and 86.2% of treated patients with AA (rs7270101) and CC genotypes (rs1127354), respectively. Men with AA (rs7270101) showed a considerable reduction in Hb at week 12 compared to those with AC/CC (p = 0.1475). In women, there was no influence of genotype (p = 0.5295). For rs1127354, men with the CC genotype also showed a sudden reduction in Hb compared to those with AC. Allelic distribution of rs7270101 and rs1127354 shows high rates of the genotypes AA and CC, respectively, suggesting that the study population had a great propensity for developing RBV-induced anaemia. A progressive Hb reduction during treatment was observed; however, this reduction was greater in men at week 12 than in women. PMID:26154744

  14. Adenosine gates synaptic plasticity at hippocampal mossy fiber synapses

    Moore, Kimberly A.; Nicoll, Roger A.; Schmitz, Dietmar


    The release properties of synapses in the central nervous system vary greatly, not only across anatomically distinct types of synapses but also among the same class of synapse. This variation manifests itself in large part by differences in the probability of transmitter release, which affects such activity-dependent presynaptic forms of plasticity as paired-pulse facilitation and frequency facilitation. This heterogeneity in presynaptic function reflects differences in the intrinsic properties of the synaptic terminal and the activation of presynaptic neurotransmitter receptors. Here we show that the unique presynaptic properties of the hippocampal mossy fiber synapse are largely imparted onto the synapse by the continuous local action of extracellular adenosine at presynaptic A1 adenosine receptors, which maintains a low basal probability of transmitter release.

  15. Crystal Structure of Schistosoma mansoni Adenosine Phosphorylase/5’-Methylthioadenosine Phosphorylase and Its Importance on Adenosine Salvage Pathway

    Torini, Juliana Roberta; Brandão-Neto, José; DeMarco, Ricardo; Pereira, Humberto D'Muniz


    Schistosoma mansoni do not have de novo purine pathways and rely on purine salvage for their purine supply. It has been demonstrated that, unlike humans, the S. mansoni is able to produce adenine directly from adenosine, although the enzyme responsible for this activity was unknown. In the present work we show that S. mansoni 5´-deoxy-5´-methylthioadenosine phosphorylase (MTAP, E.C. is capable of use adenosine as a substrate to the production of adenine. Through kinetics assays, we show that the Schistosoma mansoni MTAP (SmMTAP), unlike the mammalian MTAP, uses adenosine substrate with the same efficiency as MTA phosphorolysis, which suggests that this enzyme is part of the purine pathway salvage in S. mansoni and could be a promising target for anti-schistosoma therapies. Here, we present 13 SmMTAP structures from the wild type (WT), including three single and one double mutant, and generate a solid structural framework for structure description. These crystal structures of SmMTAP reveal that the active site contains three substitutions within and near the active site when compared to it mammalian counterpart, thus opening up the possibility of developing specific inhibitors to the parasite MTAP. The structural and kinetic data for 5 substrates reveal the structural basis for this interaction, providing substract for inteligent design of new compounds for block this enzyme activity. PMID:27935959

  16. Repeated administration of adenosine increases its cardiovascular effects in rats.

    Vidrio, H; García-Márquez, F; Magos, G A


    Hypotensive and negative chronotropic responses to adenosine in anesthetized rats increased after previous administration of the nucleoside. Bradycardia after adenosine in the isolated perfused rat heart was also potentiated after repeated administration at short intervals. This self-potentiation could be due to extracellular accumulation of adenosine and persistent stimulation of receptors caused by saturation or inhibition of cellular uptake of adenosine.

  17. NTS adenosine A2a receptors inhibit the cardiopulmonary chemoreflex control of regional sympathetic outputs via a GABAergic mechanism.

    Minic, Zeljka; O'Leary, Donal S; Scislo, Tadeusz J


    Adenosine is a powerful central neuromodulator acting via opposing A1 (inhibitor) and A2a (activator) receptors. However, in the nucleus of the solitary tract (NTS), both adenosine receptor subtypes attenuate cardiopulmonary chemoreflex (CCR) sympathoinhibition of renal, adrenal, and lumbar sympathetic nerve activity and attenuate reflex decreases in arterial pressure and heart rate. Adenosine A1 receptors inhibit glutamatergic transmission in the CCR pathway, whereas adenosine A2a receptors most likely facilitate release of an unknown inhibitory neurotransmitter, which, in turn, inhibits the CCR. We hypothesized that adenosine A2a receptors inhibit the CCR via facilitation of GABA release in the NTS. In urethane-chloralose-anesthetized rats (n = 51), we compared regional sympathetic responses evoked by stimulation of the CCR with right atrial injections of the 5-HT3 receptor agonist phenylbiguanide (1-8 μg/kg) before and after selective stimulation of NTS adenosine A2a receptors [microinjections into the NTS of CGS-21680 (20 pmol/50 nl)] preceded by blockade of GABAA or GABAB receptors in the NTS [bicuculline (10 pmol/100 nl) or SCH-50911 (1 nmol/100 nl)]. Blockade of GABAA receptors virtually abolished adenosine A2a receptor-mediated inhibition of the CCR. GABAB receptors had much weaker but significant effects. These effects were similar for the different sympathetic outputs. We conclude that stimulation of NTS adenosine A2a receptors inhibits CCR-evoked hemodynamic and regional sympathetic reflex responses via a GABA-ergic mechanism.

  18. Involvement of adenosine A2a receptor in intraocular pressure decrease induced by 2-(1-octyn-1-yl)adenosine or 2-(6-cyano-1-hexyn-1-yl)adenosine.

    Konno, Takashi; Murakami, Akira; Uchibori, Takehiro; Nagai, Akihiko; Kogi, Kentaro; Nakahata, Norimichi


    The aim of the present study is to clarify the mechanism for the decrease in intraocular pressure by 2-alkynyladenosine derivatives in rabbits. The receptor binding analysis revealed that 2-(1-octyn-1-yl)adenosine (2-O-Ado) and 2-(6-cyano-1-hexyn-1-yl)adenosine (2-CN-Ado) selectively bound to the A(2a) receptor with a high affinity. Ocular hypotensive responses to 2-O-Ado and 2-CN-Ado were inhibited by the adenosine A(2a)-receptor antagonist 1,3,7-trimethyl-8-(3-chlorostyryl)xanthine (CSC), but not by the adenosine A(1)-receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) or the adenosine A(2b)-receptor antagonist alloxazine. In addition, 2-O-Ado and 2-CN-Ado caused an increase in outflow facility, which was inhibited by CSC, but not by DPCPX or alloxazine. Moreover, 2-O-Ado and 2-CN-Ado increased cAMP in the aqueous humor, and the 2-O-Ado-induced an increase in cAMP was inhibited by CSC. These results suggest that 2-O-Ado and 2-CN-Ado reduced intraocular pressure via an increase in outflow facility. The ocular hypotension may be mainly mediated through the activation of adenosine A(2a) receptor, although a possible involvement of adenosine A(1) receptor cannot be completely ruled out. 2-O-Ado and 2-CN-Ado are useful lead compounds for the treatment of glaucoma.

  19. Overexpression of human selenoprotein H in neuronal cells enhances mitochondrial biogenesis and function through activation of protein kinase A, protein kinase B, and cyclic adenosine monophosphate response element-binding protein pathway.

    Mehta, Suresh L; Mendelev, Natalia; Kumari, Santosh; Andy Li, P


    Mitochondrial biogenesis is activated by nuclear encoded transcription co-activator peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), which is regulated by several upstream factors including protein kinase A and Akt/protein kinase B. We have previously shown that selenoprotein H enhances the levels of nuclear regulators for mitochondrial biogenesis, increases mitochondrial mass and improves mitochondrial respiratory rate, under physiological condition. Furthermore, overexpression of selenoprotein H protects neuronal HT22 cells from ultraviolet B irradiation-induced cell damage by lowering reactive oxygen species production, and inhibiting activation of caspase-3 and -9, as well as p53. The objective of this study is to identify the cell signaling pathways by which selenoprotein H initiates mitochondrial biogenesis. We first confirmed our previous observation that selenoprotein H transfected HT22 cells increased the protein levels of nuclear-encoded mitochondrial biogenesis factors, peroxisome proliferator-activated receptor γ coactivator-1α, nuclear respiratory factor 1 and mitochondrial transcription factor A. We then observed that total and phosphorylation of protein kinase A, Akt/protein kinase B and cyclic adenosine monophosphate response element-binding protein (CREB) were significantly increased in selenoprotein H transfected cells compared to vector transfected HT22 cells. To verify whether the observed stimulating effects on mitochondrial biogenesis pathways are caused by selenoprotein H and mediated through CREB, we knocked down selenoprotein H mRNA level using siRNA and inhibited CREB with napthol AS-E phosphate in selenoprotein H transfected cells and repeated the measurements of the aforementioned biomarkers. Our results revealed that silencing of selenoprotein H not only decreased the protein levels of PGC-1α, nuclear respiratory factor 1 and mitochondrial transcription factor A, but also decreased the total and

  20. Adenosine receptor control of cognition in normal and disease.

    Chen, Jiang-Fan


    Adenosine and adenosine receptors (ARs) are increasingly recognized as important therapeutic targets for controlling cognition under normal and disease conditions for its dual roles of neuromodulation as well as of homeostatic function in the brain. This chapter first presents the unique ability of adenosine, by acting on the inhibitory A1 and facilitating A2A receptor, to integrate dopamine, glutamate, and BNDF signaling and to modulate synaptic plasticity (e.g., long-term potentiation and long-term depression) in brain regions relevant to learning and memory, providing the molecular and cellular bases for adenosine receptor (AR) control of cognition. This led to the demonstration of AR modulation of social recognition memory, working memory, reference memory, reversal learning, goal-directed behavior/habit formation, Pavlovian fear conditioning, and effort-related behavior. Furthermore, human and animal studies support that AR activity can also, through cognitive enhancement and neuroprotection, reverse cognitive impairments in animal models of Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease, and schizophrenia. Lastly, epidemiological evidence indicates that regular human consumption of caffeine, the most widely used psychoactive drug and nonselective AR antagonists, is associated with the reduced cognitive decline in aging and AD patients, and with the reduced risk in developing PD. Thus, there is a convergence of the molecular studies revealing AR as molecular targets for integrating neurotransmitter signaling and controlling synaptic plasticity, with animal studies demonstrating the strong procognitive impact upon AR antagonism in normal and disease brains and with epidemiological and clinical evidences in support of caffeine and AR drugs for therapeutic modulation of cognition. Since some of adenosine A2A receptor antagonists are already in phase III clinical trials for motor benefits in PD patients with remarkable safety profiles

  1. Increased Na+/K(+)-pump activity and adenosine triphosphate utilization after compound 48/80-induced histamine secretion from rat mast cells

    Johansen, Torben; Praetorius, Birger Hans


    -production were measured by the bioluminescence technique (firefly lantern) and by measurement of the lactate production under anaerobic conditions (antimycin A, oligomycin), respectively. There was an increased requirement for ATP after the secretory response associated with an increased activity of the Na...

  2. Isoform-specific regulation of the Na+-K+ pump by adenosine in guinea pig ventricular myocytes

    Zhe ZHANG; Hui-cai GUO; Li-nan ZHANG; Yong-li WANG


    Aim: The present study investigated the effect of adenosine on Na+-K+ pumps in acutely isolated guinea pig (C, avia sp.) ven-tricular myocytes.Methods: The whole-cell, patch-damp technique was used to record the Na+-K+ pump current (Ip) in acutely isolated guinea pig ventricular myocytes.Results: Adenosine inhibited the high DHO-affinity pump current (Ih) in a concentration-dependent manner, which was blocked by the selective adenosine A1 receptor antagonist DPCPX and the general protein kinase C (PKC) antagonists stau-rosporine, GF 109203X or the specific δ isoform antagonist rottlerin. In addition, the inhibitory action of adenosine was mimicked by a selective A1 receptor agonist CCPA and a specific activator peptide of PKC-δ, PP114. In contrast, the selec-tive A2A receptor agonist CGS21680 and A3 receptor agonist Cl-IB-MECA did not affect lb. Application of the selective A2A receptor antagonist SCH58261 and A3 receptor antagonist MRS1191 also failed to block the effect of adenosine. Further-more, H89, a selective protein kinase A (PKA) antagonist, did not exert any effect on adenosine-induced Ih inhibition.Conclusion: The present study provides the electrophysiological evidence that adenosine can induce significant inhibition of Ih via adenosine A1 receptors and the PKC-δ isoform.

  3. Caffeine stimulates locomotor activity in the mammalian spinal cord via adenosine A1 receptor-dopamine D1 receptor interaction and PKA-dependent mechanisms.

    Acevedo, JeanMarie; Santana-Almansa, Alexandra; Matos-Vergara, Nikol; Marrero-Cordero, Luis René; Cabezas-Bou, Ernesto; Díaz-Ríos, Manuel


    Caffeine is a potent psychostimulant that can have significant and widely variable effects on the activity of multiple neuronal pathways. The most pronounced caffeine-induced behavioral effect seen in rodents is to increase locomotor activity which has been linked to a dose-dependent inhibition of A1 and A(2A) receptors. The effects of caffeine at the level of the lumbar spinal central pattern generator (CPG) network for hindlimb locomotion are lacking. We assessed the effects of caffeine to the locomotor function of the spinal CPG network via extracellular ventral root recordings using the isolated neonatal mouse spinal cord preparation. Addition of caffeine and of an A1 receptor antagonist significantly decreased the cycle period accelerating the ongoing locomotor rhythm, while decreasing burst duration reversibly in most preparations suggesting the role of A1 receptors as the primary target of caffeine. Caffeine and an A1 receptor antagonist failed to stimulate ongoing locomotor activity in the absence of dopamine or in the presence of a D1 receptor antagonist supporting A1/D1 receptor-dependent mechanism of action. The use of caffeine or an A1 receptor blocker failed to stimulate an ongoing locomotor rhythm in the presence of a blocker of the cAMP-dependent protein kinase (PKA) supporting the need of this intracellular pathway for the modulatory effects of caffeine to occur. These results support a stimulant effect of caffeine on the lumbar spinal network controlling hindlimb locomotion through the inhibition of A1 receptors and subsequent activation of D1 receptors via a PKA-dependent intracellular mechanism.

  4. Cyclic 3'-5'-adenosine monophosphate binds to annexin I and regulates calcium-dependent membrane aggregation and ion channel activity.

    Cohen, B E; Lee, G; Arispe, N; Pollard, H B


    The annexin (Anx) gene family comprises a set of calcium-dependent membrane binding proteins, which have been implicated in a wide variety of cellular processes including membrane fusion and calcium channel activity. We report here that cAMP activates Ca(2+)-dependent aggregation of both phosphatidylserine (PS) liposomes and bovine chromaffin granules driven by [des 1-12]annexin I (lipocortin I, Anx1). The mechanism of cAMP action involves an increase in AnxI-dependent cooperativity on the rate of such a reaction without affecting the corresponding k1/2 values. Cyclic AMP causes the values of the Hill coefficient (nH) for AnxI to change from 3 to 6 in both PS liposomes and chromaffin granules. By contrast, ATP inhibits the rate of aggregation activity without affecting the cooperativity or the extent of aggregation process. We were also able to photolabel Anx1 specifically with an 8-azido analogue of cAMP by a calcium-independent process. Such a process is saturable, yielding a Kd = 0.8 microM by Scatchard analysis. Specific displacement occurs in the presence of cAMP and ATP. Finally, we found that cAMP alters the conductance of calcium channels formed by AnxI in planar lipid bilayers. We interpret these data to indicate that AnxI binds both calcium and cAMP independently, and that both actions have functional consequences. This is the first report of a nucleotide binding function for a member of the annexin gene family.

  5. In vivo effects of adenosine 5´-triphosphate on rat preneoplastic liver

    Ana V. Frontini


    Full Text Available The utilization of adenosine 5´-triphosphate (ATP infusions to inhibit the growth of some human and animals tumors was based on the anticancer activity observed in in vitro and in vivo experiments, but contradictory results make the use of ATP in clinical practice rather controversial. Moreover, there is no literature regarding the use of ATP infusions to treat hepatocarcinomas. The purpose of this study was to investigate whether ATP prevents in vivo oncogenesis in very-early-stage cancer cells in a well characterized two-stage model of hepatocarcinogenesis in the rat. As we could not preclude the possible effect due to the intrinsic properties of adenosine, a known tumorigenic product of ATP hydrolysis, the effect of the administration of adenosine was also studied. Animals were divided in groups: rats submitted to the two stage preneoplasia initiation/promotion model of hepatocarcinogenesis, rats treated with intraperitoneal ATP or adenosine during the two phases of the model and appropriate control groups. The number and volume of preneoplastic foci per liver identified by the expression of glutathione S-transferase placental type and the number of proliferating nuclear antigen positive cells significantly increased in ATP and adenosine treated groups. Taken together, these results indicate that in this preneoplastic liver model, ATP as well as adenosine disturb the balance between apoptosis and proliferation contributing to malignant transformation.

  6. Uridine adenosine tetraphosphate (Up{sub 4}A) is a strong inductor of smooth muscle cell migration via activation of the P2Y{sub 2} receptor and cross-communication to the PDGF receptor

    Wiedon, Annette; Toelle, Markus; Bastine, Joschika; Schuchardt, Mirjam; Huang, Tao; Jankowski, Vera; Jankowski, Joachim; Zidek, Walter [Charite - Universitaetsmedizin Berlin, ChariteCentrum 13, Department of Nephrology, Campus Benjamin Franklin, Hindenburgdamm 30, 12203 Berlin (Germany); Giet, Markus van der, E-mail: [Charite - Universitaetsmedizin Berlin, ChariteCentrum 13, Department of Nephrology, Campus Benjamin Franklin, Hindenburgdamm 30, 12203 Berlin (Germany)


    Highlights: Black-Right-Pointing-Pointer Up{sub 4}A induces VSMC migration. Black-Right-Pointing-Pointer VSMC migration towards Up{sub 4}A involves P2Y{sub 2} activation. Black-Right-Pointing-Pointer Up{sub 4}A-induced VSMC migration is OPN-dependent. Black-Right-Pointing-Pointer Activation of ERK1/2 pathway is necessary for VSMC migration towards Up{sub 4}A. Black-Right-Pointing-Pointer Up{sub 4}A-directed VSMC migration cross-communicates with the PDGFR. -- Abstract: The recently discovered dinucleotide uridine adenosine tetraphosphate (Up{sub 4}A) was found in human plasma and characterized as endothelium-derived vasoconstrictive factor (EDCF). A further study revealed a positive correlation between Up{sub 4}A and vascular smooth muscle cell (VSMC) proliferation. Due to the dominant role of migration in the formation of atherosclerotic lesions our aim was to investigate the migration stimulating potential of Up{sub 4}A. Indeed, we found a strong chemoattractant effect of Up{sub 4}A on VSMC by using a modified Boyden chamber. This migration dramatically depends on osteopontin secretion (OPN) revealed by the reduction of the migration signal down to 23% during simultaneous incubation with an OPN-blocking antibody. Due to inhibitory patterns using specific and unspecific purinoreceptor inhibitors, Up{sub 4}A mediates it's migratory signal mainly via the P2Y{sub 2}. The signaling behind the receptor was investigated with luminex technique and revealed an activation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathway. By use of the specific PDGF receptor (PDGFR) inhibitor AG1296 and siRNA technique against PDGFR-{beta} we found a strongly reduced migration signal after Up{sub 4}A stimulation in the PDGFR-{beta} knockdown cells compared to control cells. In this study, we present substantiate data that Up{sub 4}A exhibits migration stimulating potential probably involving the signaling cascade of MEK1 and ERK1/2 as well as the matrix protein

  7. Post-Meal Responses of Elongation Factor 2 (eEF2 and Adenosine Monophosphate-Activated Protein Kinase (AMPK to Leucine and Carbohydrate Supplements for Regulating Protein Synthesis Duration and Energy Homeostasis in Rat Skeletal Muscle

    Donald K. Layman


    Full Text Available Previous research demonstrates that the anabolic response of muscle protein synthesis (MPS to a meal is regulated at the level of translation initiation with signals derived from leucine (Leu and insulin to activate mTORC1 signaling. Recent evidence suggests that the duration of the meal response is limited by energy status of the cell and inhibition of translation elongation factor 2 (eEF2. This study evaluates the potential to extend the anabolic meal response with post-meal supplements of Leu or carbohydrates. Adult (~256 g male Sprague-Dawley rats were food deprived for 12 h, then either euthanized before a standard meal (time 0 or at 90 or 180 min post-meal. At 135 min post-meal, rats received one of five oral supplements: 270 mg leucine (Leu270, 80:40:40 mg leucine, isoleucine, and valine (Leu80, 2.63 g carbohydrates (CHO2.6, 1 g carbohydrates (CHO1.0, or water (Sham control. Following the standard meal, MPS increased at 90 min then declined to pre-meal baseline at 180 min. Rats administered Leu270, Leu80, CHO2.6, or CHO1.0 maintained elevated rates of MPS at 180 min, while Sham controls declined from peak values. Leu80 and CHO1.0 treatments maintained MPS, but with values intermediate between Sham controls and Leu270 and CHO2.6 supplements. Consistent with MPS findings, the supplements maintained elongation activity and cellular energy status by preventing increases in AMP/ATP and phosphorylation of adenosine monophosphate-activated protein kinase (AMPK, acetyl-CoA carboxylase ACC and eEF2. The impact of the supplements on MPS and cellular energy status was in proportion to the energy content within the individual treatments (i.e., Leu270 > Leu80; CHO2.6 > CHO1.0, but the Leu supplements produced a disproportionate anabolic stimulation of MPS, eEF2 and energy status with significantly lower energy content. In summary, the incongruity between MPS and translation initiation at 180 min reflects a block in translation elongation due to

  8. Post-meal responses of elongation factor 2 (eEF2) and adenosine monophosphate-activated protein kinase (AMPK) to leucine and carbohydrate supplements for regulating protein synthesis duration and energy homeostasis in rat skeletal muscle.

    Wilson, Gabriel J; Moulton, Christopher J; Garlick, Peter J; Anthony, Tracy G; Layman, Donald K


    Previous research demonstrates that the anabolic response of muscle protein synthesis (MPS) to a meal is regulated at the level of translation initiation with signals derived from leucine (Leu) and insulin to activate mTORC1 signaling. Recent evidence suggests that the duration of the meal response is limited by energy status of the cell and inhibition of translation elongation factor 2 (eEF2). This study evaluates the potential to extend the anabolic meal response with post-meal supplements of Leu or carbohydrates. Adult (~256 g) male Sprague-Dawley rats were food deprived for 12 h, then either euthanized before a standard meal (time 0) or at 90 or 180 min post-meal. At 135 min post-meal, rats received one of five oral supplements: 270 mg leucine (Leu270), 80:40:40 mg leucine, isoleucine, and valine (Leu80), 2.63 g carbohydrates (CHO2.6), 1 g carbohydrates (CHO1.0), or water (Sham control). Following the standard meal, MPS increased at 90 min then declined to pre-meal baseline at 180 min. Rats administered Leu270, Leu80, CHO2.6, or CHO1.0 maintained elevated rates of MPS at 180 min, while Sham controls declined from peak values. Leu80 and CHO1.0 treatments maintained MPS, but with values intermediate between Sham controls and Leu270 and CHO2.6 supplements. Consistent with MPS findings, the supplements maintained elongation activity and cellular energy status by preventing increases in AMP/ATP and phosphorylation of adenosine monophosphate-activated protein kinase (AMPK), acetyl-CoA carboxylase ACC and eEF2. The impact of the supplements on MPS and cellular energy status was in proportion to the energy content within the individual treatments (i.e., Leu270 > Leu80; CHO2.6 > CHO1.0), but the Leu supplements produced a disproportionate anabolic stimulation of MPS, eEF2 and energy status with significantly lower energy content. In summary, the incongruity between MPS and translation initiation at 180 min reflects a block in translation elongation due to reduced

  9. Adenosine derived from Staphylococcus aureus-engulfed macrophages functions as a potent stimulant for the induction of inflammatory cytokines in mast cells

    Ma, Ying Jie; Kim, Chan-Hee; Ryu, Kyoung-Hwa;


    In this study, we attempted to isolate novel mast cell-stimulating molecules from Staphylococcus aureus. Water-soluble extract of S. aureus cell lysate strongly induced human interleukin- 8 in human mast cell line-1 and mouse interleukin-6 in mouse bone marrow-derived mast cells. The active...... adenosine receptor blocker, verified that purified adenosine can induce interleukin-8 production via adenosine receptors on mast cells. Moreover, adenosine was purified from S. aureusengulfed RAW264.7 cells, a murine macrophage cell line, used to induce phagocytosis of S. aureus. These results show a novel...

  10. Modulation and metamodulation of synapses by adenosine.

    Ribeiro, J A; Sebastião, A M


    The presence of adenosine in all nervous system cells (neurones and glia) together with its intensive release following insults makes adenosine as a sort of 'regulator' of synaptic communication, leading to the homeostatic coordination of brain function. Besides the direct actions of adenosine on the neurosecretory mechanisms, to tune neurotransmitter release, adenosine receptors interact with other receptors as well as with transporters as part of its attempt to fine-tune synaptic transmission. This review will focus on examples of the different ways adenosine can use to modulate or metamodulate synapses, in other words, to trigger or brake the action of some neurotransmitters and neuromodulators, to cross-talk with other G protein-coupled receptors, with ionotropic receptors and with receptor kinases as well as with transporters. Most of these interactions occur through A2A receptors, which in spite of their low density in some brain areas, such as the hippocampus, may function as amplifiers of the signalling of other mediators at synapses.

  11. Role of adenosine in the antiepileptic effects of deep brain stimulation

    Miranda, Maisa F.; Hamani, Clement; de Almeida, Antônio-Carlos G.; Amorim, Beatriz O.; Macedo, Carlos E.; Fernandes, Maria José S.; Nobrega, José N.; Aarão, Mayra C.; Madureira, Ana Paula; Rodrigues, Antônio M.; Andersen, Monica L.; Tufik, Sergio; Mello, Luiz E.; Covolan, Luciene


    Despite the effectiveness of anterior thalamic nucleus (AN) deep brain stimulation (DBS) for the treatment of epilepsy, mechanisms responsible for the antiepileptic effects of this therapy remain elusive. As adenosine modulates neuronal excitability and seizure activity in animal models, we hypothesized that this nucleoside could be one of the substrates involved in the effects of AN DBS. We applied 5 days of stimulation to rats rendered chronically epileptic by pilocarpine injections and recorded epileptiform activity in hippocampal slices. We found that slices from animals given DBS had reduced hippocampal excitability and were less susceptible to develop ictal activity. In live animals, AN DBS significantly increased adenosine levels in the hippocampus as measured by microdialysis. The reduced excitability of DBS in vitro was completely abolished in animals pre-treated with A1 receptor antagonists and was strongly potentiated by A1 receptor agonists. We conclude that some of the antiepileptic effects of DBS may be mediated by adenosine. PMID:25324724

  12. Vaccinia virus lacking the deoxyuridine triphosphatase gene (F2L replicates well in vitro and in vivo, but is hypersensitive to the antiviral drug (N-methanocarbathymidine

    Moyer Richard W


    Full Text Available Abstract Background The vaccinia virus (VV F2L gene encodes a functional deoxyuridine triphosphatase (dUTPase that catalyzes the conversion of dUTP to dUMP and is thought to minimize the incorporation of deoxyuridine residues into the viral genome. Previous studies with with a complex, multigene deletion in this virus suggested that the gene was not required for viral replication, but the impact of deleting this gene alone has not been determined in vitro or in vivo. Although the crystal structure for this enzyme has been determined, its potential as a target for antiviral therapy is unclear. Results The F2L gene was replaced with GFP in the WR strain of VV to assess its effect on viral replication. The resulting virus replicated well in cell culture and its replication kinetics were almost indistinguishable from those of the wt virus and attained similar titers. The virus also appeared to be as pathogenic as the WR strain suggesting that it also replicated well in mice. Cells infected with the dUTPase mutant would be predicted to affect pyrimidine deoxynucleotide pools and might be expected to exhibit altered susceptibility to pyrimidine analogs. The antiviral activity of cidofovir and four thymidine analogs were evaluated both in the mutant and the parent strain of this virus. The dUTPase knockout remained fully susceptible to cidofovir and idoxuridine, but was hypersensitive to the drug (N-methanocarbathymidine, suggesting that pyrimidine metabolism was altered in cells infected with the mutant virus. The absence of dUTPase should reduce cellular dUMP pools and may result in a reduced conversion to dTMP by thymidylate synthetase or an increased reliance on the salvage of thymidine by the viral thymidine kinase. Conclusion We confirmed that F2L was not required for replication in cell culture and determined that it does not play a significant role on virulence of the virus in intranasally infected mice. The recombinant virus is hypersensitive

  13. The role of adenosine receptors and endogenous adenosine in citalopram-induced cardiovascular toxicity

    Kubilay Oransay


    Full Text Available Aim: We investigated the role of adenosine in citalopram-induced cardiotoxicity. Materials and Methods: Protocol 1: Rats were randomized into four groups. Sodium cromoglycate was administered to rats. Citalopram was infused after the 5% dextrose, 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX; A 1 receptor antagonist, 8-(-3-chlorostyryl-caffeine (CSC; A 2a receptor antagonist, or dimethyl sulfoxide (DMSO administrations. Protocol 2: First group received 5% dextrose intraperitoneally 1 hour prior to citalopram. Other rats were pretreated with erythro-9-(2-hydroxy-3-nonyl adenine (EHNA; inhibitor of adenosine deaminase and S-(4-Nitrobenzyl-6-thioinosine (NBTI; inhibitor of facilitated adenosine transport. After pretreatment, group 2 received 5% dextrose and group 3 received citalopram. Adenosine concentrations, mean arterial pressure (MAP, heart rate (HR,  QRS duration and QT interval were evaluated. Results: In the dextrose group, citalopram infusion caused a significant decrease in MAP and HR and caused a significant prolongation in QRS and QT. DPCPX infusion significantly prevented the prolongation of the QT interval when compared to control. In the second protocol, citalopram infusion did not cause a significant change in plasma adenosine concentrations, but a significant increase observed in EHNA/NBTI groups. In EHNA/NBTI groups, citalopram-induced MAP and HR reductions, QRS and QT prolongations were more significant than the dextrose group. Conclusions: Citalopram may lead to QT prolongation by stimulating adenosine A 1 receptors without affecting the release of adenosine.

  14. 2'-O methylation of internal adenosine by flavivirus NS5 methyltransferase.

    Hongping Dong

    Full Text Available RNA modification plays an important role in modulating host-pathogen interaction. Flavivirus NS5 protein encodes N-7 and 2'-O methyltransferase activities that are required for the formation of 5' type I cap (m(7GpppAm of viral RNA genome. Here we reported, for the first time, that flavivirus NS5 has a novel internal RNA methylation activity. Recombinant NS5 proteins of West Nile virus and Dengue virus (serotype 4; DENV-4 specifically methylates polyA, but not polyG, polyC, or polyU, indicating that the methylation occurs at adenosine residue. RNAs with internal adenosines substituted with 2'-O-methyladenosines are not active substrates for internal methylation, whereas RNAs with adenosines substituted with N⁶-methyladenosines can be efficiently methylated, suggesting that the internal methylation occurs at the 2'-OH position of adenosine. Mass spectroscopic analysis further demonstrated that the internal methylation product is 2'-O-methyladenosine. Importantly, genomic RNA purified from DENV virion contains 2'-O-methyladenosine. The 2'-O methylation of internal adenosine does not require specific RNA sequence since recombinant methyltransferase of DENV-4 can efficiently methylate RNAs spanning different regions of viral genome, host ribosomal RNAs, and polyA. Structure-based mutagenesis results indicate that K61-D146-K181-E217 tetrad of DENV-4 methyltransferase forms the active site of internal methylation activity; in addition, distinct residues within the methyl donor (S-adenosyl-L-methionine pocket, GTP pocket, and RNA-binding site are critical for the internal methylation activity. Functional analysis using flavivirus replicon and genome-length RNAs showed that internal methylation attenuated viral RNA translation and replication. Polymerase assay revealed that internal 2'-O-methyladenosine reduces the efficiency of RNA elongation. Collectively, our results demonstrate that flavivirus NS5 performs 2'-O methylation of internal adenosine of

  15. Lower frequency of the low activity adenosine deaminase allelic variant (ADA1*2 in schizophrenic patients Diminuição da frequência da variante alélica de baixa atividade da adenosina desaminase (ADA1*2 em pacientes esquizofrênicos

    Gustavo Pimentel Dutra


    Full Text Available OBJECTIVE: Adenosine may play a role in the pathophysiology of schizophrenia, since it modulates the release of several neurotransmitters such as glutamate, dopamine, serotonin and acetylcholine, decreases neuronal activity by pos-synaptic hyperpolarization and inhibits dopaminergic activity. Adenosine deaminase participates in purine metabolism by converting adenosine into inosine. The most frequent functional polymorphism of adenosine deaminase (22G→A (ADA1*2 exhibits 20-30% lower enzymatic activity in individuals with the G/A genotype than individuals with the G/G genotype. The aim of this study was to evaluate the ADA polymorphism 22G→A (ADA1*2 in schizophrenic patients and healthy controls. METHOD: The genotypes of the ADA 22G→A were identified with allele-specific PCR strategy in 152 schizophrenic patients and 111 healthy individuals. RESULTS: A significant decrease in the frequency of the G/A genotype was seen in schizophrenic patients (7/152 - 4.6% relative to controls (13/111 - 11.7%, p = 0.032, OR = 2.6. CONCLUSION: These results suggest that the G/A genotype associated with low adenosine deaminase activity and, supposingly, with higher adenosine levels is less frequent among schizophrenic patients.OBJETIVO: A adenosina pode ter um papel importante na fisiopatologia da esquizofrenia, uma vez que modula a liberação de vários neurotransmissores, tais como glutamato, dopamina, serotonina e acetilcolina, diminui a atividade neuronal por hiperpolarização pós-sináptica e inibe a atividade dopaminérgica. A adenosina desaminase participa do metabolismo das purinas pela conversão de adenosina em inosina. O mais frequente polimorfismo funcional da adenosina desaminase (22G →A (ADA1*2 exibe uma diminuição de 20-30% da atividade funcional em indivíduos com genótipo G/A quando comparados com indivíduos com o genótipo G/G. O objetivo deste estudo foi avaliar o polimorfismo 22G→A (ADA1*2 em pacientes esquizofrênicos e em

  16. Bradykinin and adenosine receptors mediate desflurane induced postconditioning in human myocardium: role of reactive oxygen species

    Gérard Jean-Louis


    Full Text Available Abstract Background Desflurane during early reperfusion has been shown to postcondition human myocardium, in vitro. We investigated the role of adenosine and bradykinin receptors, and generation of radical oxygen species in desflurane-induced postconditioning in human myocardium. Methods We recorded isometric contraction of human right atrial trabeculae hanged in an oxygenated Tyrode's solution (34 degrees Celsius, stimulation frequency 1 Hz. After a 30-min hypoxic period, desflurane 6% was administered during the first 5 min of reoxygenation. Desflurane was administered alone or with pretreatment of N-mercaptopropionylglycine, a reactive oxygen species scavenger, 8-(p-Sulfophenyltheophylline, an adenosine receptor antagonist, HOE140, a selective B2 bradykinin receptor antagonist. In separate groups, adenosine and bradykinin were administered during the first minutes of reoxygenation alone or in presence of N-mercaptopropionylglycine. The force of contraction of trabeculae was recorded continuously. Developed force at the end of a 60-min reoxygenation period was compared (mean ± standard deviation between the groups by a variance analysis and post hoc test. Results Desflurane 6% (84 ± 6% of baseline enhanced the recovery of force after 60-min of reoxygenation as compared to control group (51 ± 8% of baseline, P N-mercaptopropionylglycine (54 ± 3% of baseline, 8-(p-Sulfophenyltheophylline (62 ± 9% of baseline, HOE140 (58 ± 6% of baseline abolished desflurane-induced postconditioning. Adenosine (80 ± 9% of baseline and bradykinin (83 ± 4% of baseline induced postconditioning (P vs control, N-mercaptopropionylglycine abolished the beneficial effects of adenosine and bradykinin (54 ± 8 and 58 ± 5% of baseline, respectively. Conclusions In vitro, desflurane-induced postconditioning depends on reactive oxygen species production, activation of adenosine and bradykinin B2 receptors. And, the cardioprotective effect of adenosine and bradykinin

  17. Adenosine triphosphate (ATP) as a possible indicator of extraterrestrial biology

    Chappelle, E. W.; Picciolo, G. L.


    The ubiquity of adenosine triphosphate (ATP) in terrestrial organisms provides the basis for proposing the assay of this vital metabolic intermediate for detecting extraterrestrial biological activity. If an organic carbon chemistry is present on the planets, the occurrence of ATP is possible either from biosynthetic or purely chemical reactions. However, ATP's relative complexity minimizes the probability of abiogenic synthesis. A sensitive technique for the quantitative detection of ATP was developed using the firefly bioluminescent reaction. The procedure was used successfully for the determination of the ATP content of soil and bacteria. This technique is also being investigated from the standpoint of its application in clinical medicine.

  18. Novel trypanocidal analogs of 5'-(methylthio)-adenosine.

    Sufrin, Janice R; Spiess, Arthur J; Marasco, Canio J; Rattendi, Donna; Bacchi, Cyrus J


    The purine nucleoside 5'-deoxy-5'-(hydroxyethylthio)-adenosine (HETA) is an analog of the polyamine pathway metabolite 5'-deoxy-5'-(methylthio)-adenosine (MTA). HETA is a lead structure for the ongoing development of selectively targeted trypanocidal agents. Thirteen novel HETA analogs were synthesized and examined for their in vitro trypanocidal activities against bloodstream forms of Trypanosoma brucei brucei LAB 110 EATRO and at least one drug-resistant Trypanosoma brucei rhodesiense clinical isolate. New compounds were also assessed in a cell-free assay for their activities as substrates of trypanosome MTA phosphorylase. The most potent analog in this group was 5'-deoxy-5'-(hydroxyethylthio)-tubercidin, whose in vitro cytotoxicity (50% inhibitory concentration [IC50], 10 nM) is 45 times greater than that of HETA (IC50, 450 nM) against pentamidine-resistant clinical isolate KETRI 269. Structure-activity analyses indicate that the enzymatic cleavage of HETA analogs by trypanosome MTA phosphorylase is not an absolute requirement for trypanocidal activity. This suggests that additional biochemical mechanisms are associated with the trypanocidal effects of HETA and its analogs.

  19. Striatal adenosine-cannabinoid receptor interactions in rats over-expressing adenosine A2A receptors.

    Chiodi, Valentina; Ferrante, Antonella; Ferraro, Luca; Potenza, Rosa Luisa; Armida, Monica; Beggiato, Sarah; Pèzzola, Antonella; Bader, Michael; Fuxe, Kjell; Popoli, Patrizia; Domenici, Maria Rosaria


    Adenosine A2A receptors (A2 A Rs) and cannabinoid CB1 receptors (CB1 Rs) are highly expressed in the striatum, where they functionally interact and form A2A /CB1 heteroreceptor complexes. We investigated the effects of CB1 R stimulation in a transgenic rat strain over-expressing A2 A Rs under the control of the neural-specific enolase promoter (NSEA2A rats) and in age-matched wild-type (WT) animals. The effects of the CB1 R agonist WIN 55,212-2 (WIN) were significantly lower in NSEA2A rats than in WT animals, as demonstrated by i) electrophysiological recordings of synaptic transmission in corticostriatal slices; ii) the measurement of glutamate outflow from striatal synaptosomes and iii) in vivo experiments on locomotor activity. Moreover, while the effects of WIN were modulated by both A2 A R agonist (CGS 21680) and antagonists (ZM 241385, KW-6002 and SCH-442416) in WT animals, the A2 A R antagonists failed to influence WIN-mediated effects in NSEA2A rats. The present results demonstrate that in rats with genetic neuronal over-expression of A2 A Rs, the effects mediated by CB1 R activation in the striatum are significantly reduced, suggesting a change in the stoichiometry of A2A and CB1 receptors and providing a strategy to dissect the involvement of A2 A R forming or not forming heteromers in the modulation of striatal functions. These findings add additional evidence for the existence of an interaction between striatal A2 A Rs and CB1 Rs, playing a fundamental role in the regulation of striatal functions. We studied A2A -CB1 receptor interaction in transgenic rats over-expressing adenosine A2A receptors under the control of the neuron-specific enolase promoter (NSEA2A ). In these rats, we demonstrated a reduced effect of the CB1 receptor agonist WIN 55,212-2 in the modulation of corticostriatal synaptic transmission and locomotor activity, while CB1 receptor expression level did not change with respect to WT rats. A reduction in the expression of A2A -CB1

  20. Adenosine stimulates the migration of human endothelial progenitor cells. Role of CXCR4 and microRNA-150.

    Magali Rolland-Turner

    Full Text Available BACKGROUND: Administration of endothelial progenitor cells (EPC represents a promising option to regenerate the heart after myocardial infarction, but is limited because of low recruitment and engraftment in the myocardium. Mobilization and migration of EPC are mainly controlled by stromal cell-derived factor 1α (SDF-1α and its receptor CXCR4. We hypothesized that adenosine, a cardioprotective molecule, may improve the recruitment of EPC to the heart. METHODS: EPC were obtained from peripheral blood mononuclear cells of healthy volunteers. Expression of chemokines and their receptors was evaluated using microarrays, quantitative PCR, and flow cytometry. A Boyden chamber assay was used to assess chemotaxis. Recruitment of EPC to the infarcted heart was evaluated in rats after permanent occlusion of the left anterior descending coronary artery. RESULTS: Microarray analysis revealed that adenosine modulates the expression of several members of the chemokine family in EPC. Among these, CXCR4 was up-regulated by adenosine, and this result was confirmed by quantitative PCR (3-fold increase, P<0.001. CXCR4 expression at the cell surface was also increased. This effect involved the A(2B receptor. Pretreatment of EPC with adenosine amplified their migration towards recombinant SDF-1α or conditioned medium from cardiac fibroblasts. Both effects were abolished by CXCR4 blocking antibodies. Adenosine also increased CXCR4 under ischemic conditions, and decreased miR-150 expression. Binding of miR-150 to the 3' untranslated region of CXCR4 was verified by luciferase assay. Addition of pre-miR-150 blunted the effect of adenosine on CXCR4. Administration of adenosine to rats after induction of myocardial infarction stimulated EPC recruitment to the heart and enhanced angiogenesis. CONCLUSION: Adenosine increases the migration of EPC. The mechanism involves A(2B receptor activation, decreased expression of miR-150 and increased expression of CXCR4. These

  1. Relation of Na+, K(+)-ATPase to delayed motor nerve conduction velocity: effect of aldose reductase inhibitor, ADN-138, on Na+, K(+)-ATPase activity.

    Hirata, Y; Okada, K


    The role of sorbitol, myo-inositol, and Na+, K(+)-adenosine triphosphatase (ATPase) activity on motor nerve conduction velocity (MNCV) in streptozotocin (STZ)-diabetic rats was studied. Reduction of MNCV and Na+, K(+)-ATPase in caudal nerves appeared after 3 weeks of diabetes, and at this time treatment with aldose reductase inhibitor (ARI), ADN-138 and 1% myo-inositol supplement was begun. One percent myo-inositol supplement for 3 weeks resulted in a significant increase in myo-inositol levels in diabetic nerves, but left MNCV and sorbitol levels unchanged. In contrast, treatment with ADN-138 for 3 weeks reduced sorbitol levels in diabetic nerves and resulted in significant increases in MNCV and Na+, K(+)-ATPase in the nerves. Since ADN-138 did not restore myo-inositol levels, the increase in Na+, K(+)-ATPase levels by ADN-138 treatment was independent of myo-inositol levels. Also, nerve Na+ levels in ADN-138-treated rats were reduced and the ratio of K+ to Na+ was raised, while 1% myo-inositol supplement did not affect them. These results suggest that treatment with ADN-138 elevates MNCV through a series of processes: ARI----reduction of sorbitol level----increase in Na+, K(+)-ATPase activity----correction of K+, Na+ imbalance----increase in MNCV.


    Ernster, Lars; Jones, Lois C.


    Rat liver microsomes catalyze the hydrolysis of the triphosphates of adenosine, guanosine, uridine, cytidine, and inosine into the corresponding diphosphates and inorganic orthophosphate. The activities are stimulated by Na2S2O4, and inhibited by atebrin, chlorpromazine, sodium azide, and deaminothyroxine. Sodium deoxycholate inhibits the ATPase activity in a progressive manner; the release of orthophosphate from GTP and UTP is stimulated by low, and inhibited by high, concentrations of deoxycholate, and that from CTP and ITP is unaffected by low, and inhibited by high, concentrations of deoxycholate. Subfractionation of microsomes with deoxycholate into ribosomal, membrane, and soluble fractions reveals a concentration of the triphosphatase activity in the membrane fraction. Rat liver microsomes also catalyze the hydrolysis of the diphosphates of the above nucleosides into the corresponding monophosphates and inorganic orthophosphate. Deoxycholate strongly enhances the GDPase, UDPase, and IDPase activities while causing no activation or even inhibition of the ADPase and CDPase activities. The diphosphatase is unaffected by Na2S2O4 and is inhibited by azide and deaminothyroxine but not by atebrin or chlorpromazine. Upon fractionation of the microsomes with deoxycholate, a large part of the GDPase, UDPase, and IDPase activities is recovered in the soluble fraction. Mechanical disruption of the microsomes with an Ultra Turrax Blender both activates and releases the GDPase, UDPase, and IDPase activities, and the former effect occurs more readily than the latter. The GDPase, UDPase, and IDPase activities of the rat liver cell reside almost exclusively in the microsomal fraction, as revealed by comparative assays of the mitochondrial, microsomal, and final supernatant fractions of the homogenate. The microsomes exhibit relatively low nucleoside monophosphatase and inorganic pyrophosphatase activities, and these are unaffected by deoxycholate or mechanical treatment

  3. Aberrant bone density in aging mice lacking the adenosine transporter ENT1.

    David J Hinton

    Full Text Available Adenosine is known to regulate bone production and resorption in humans and mice. Type 1 equilibrative nucleoside transporter (ENT1 is responsible for the majority of adenosine transport across the plasma membrane and is ubiquitously expressed in both humans and mice. However, the contribution of ENT1-mediated adenosine levels has not been studied in bone remodeling. With the recent identification of the importance of adenosine signaling in bone homeostasis, it is essential to understand the role of ENT1 to develop novel therapeutic compounds for bone disorders. Here we examined the effect of ENT1 deletion on bone density using X-ray, dual energy X-ray absorptiometry and micro-computerized tomography analysis. Our results show that bone density and bone mineral density is reduced in the lower thoracic and lumbar spine as well as the femur of old ENT1 null mice (>7 months compared to wild-type littermates. Furthermore, we found increased mRNA expression of tartrate-resistant acid phosphatase (TRAP, an osteoclast marker, in isolated long bones from 10 month old ENT1 null mice compared to wild-type mice. In addition, aged ENT1 null mice displayed severe deficit in motor coordination and locomotor activity, which might be attributed to dysregulated bone density. Overall, our study suggests that ENT1-regulated adenosine signaling plays an essential role in lumbar spine and femur bone density.

  4. Squalenoyl adenosine nanoparticles provide neuroprotection after stroke and spinal cord injury

    Gaudin, Alice; Yemisci, Müge; Eroglu, Hakan; Lepetre-Mouelhi, Sinda; Turkoglu, Omer Faruk; Dönmez-Demir, Buket; Caban, Seçil; Sargon, Mustafa Fevzi; Garcia-Argote, Sébastien; Pieters, Grégory; Loreau, Olivier; Rousseau, Bernard; Tagit, Oya; Hildebrandt, Niko; Le Dantec, Yannick; Mougin, Julie; Valetti, Sabrina; Chacun, Hélène; Nicolas, Valérie; Desmaële, Didier; Andrieux, Karine; Capan, Yilmaz; Dalkara, Turgay; Couvreur, Patrick


    There is an urgent need to develop new therapeutic approaches for the treatment of severe neurological trauma, such as stroke and spinal cord injuries. However, many drugs with potential neuropharmacological activity, such as adenosine, are inefficient upon systemic administration because of their fast metabolization and rapid clearance from the bloodstream. Here, we show that conjugation of adenosine to the lipid squalene and the subsequent formation of nanoassemblies allows prolonged circulation of this nucleoside, providing neuroprotection in mouse stroke and rat spinal cord injury models. The animals receiving systemic administration of squalenoyl adenosine nanoassemblies showed a significant improvement of their neurologic deficit score in the case of cerebral ischaemia, and an early motor recovery of the hindlimbs in the case of spinal cord injury. Moreover, in vitro and in vivo studies demonstrated that the nanoassemblies were able to extend adenosine circulation and its interaction with the neurovascular unit. This Article shows, for the first time, that a hydrophilic and rapidly metabolized molecule such as adenosine may become pharmacologically efficient owing to a single conjugation with the lipid squalene.

  5. Online cleanup of accelerated solvent extractions for determination of adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and adenosine 5'-monophosphate (AMP) in royal jelly using high-performance liquid chromatography.

    Xue, Xiaofeng; Wang, Feng; Zhou, Jinhui; Chen, Fang; Li, Yi; Zhao, Jing


    Determination of the levels of adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and adenosine 5'-monophosphate (AMP) in royal jelly is important for the study of its pharmacological activities, health benefits, and adenosine phosphate degradation. In this study was developed a novel method to determine ATP, ADP, and AMP levels in royal jelly using accelerated solvent extraction (ASE) followed by online cleanup and high-performance liquid chromatography (HPLC) with diode array detection (DAD). The optimum extraction conditions were obtained using an 11 mL ASE cell, ethanol/water (5:5 v/v) as the extraction solvent, 1500 psi, 80 degrees C, a 5 min static time, and a 60% flush volume. Optimum separation of the three compounds was achieved in AMP levels in 15 samples of royal jelly of different origins was performed. Sample results indicated that the AMP concentration was 24.2-2214.4 mg kg(-1), whereas ATP and ADP were not detectable or present only at low levels.

  6. A3 Adenosine receptors mediate oligodendrocyte death and ischemic damage to optic nerve.

    González-Fernández, Estíbaliz; Sánchez-Gómez, María Victoria; Pérez-Samartín, Alberto; Arellano, Rogelio O; Matute, Carlos


    Adenosine receptor activation is involved in myelination and in apoptotic pathways linked to neurodegenerative diseases. In this study, we investigated the effects of adenosine receptor activation in the viability of oligodendrocytes of the rat optic nerve. Selective activation of A3 receptors in pure cultures of oligodendrocytes caused concentration-dependent apoptotic and necrotic death which was preceded by oxidative stress and mitochondrial membrane depolarization. Oligodendrocyte apoptosis induced by A3 receptor activation was caspase-dependent and caspase-independent. In addition to dissociated cultures, incubation of optic nerves ex vivo with adenosine and the A3 receptor agonist 2-CI-IB-MECA(1-[2-Chloro-6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-b-D-ribofuranuronamide)-induced caspase-3 activation, oligodendrocyte damage, and myelin loss, effects which were prevented by the presence of caffeine and the A3 receptor antagonist MRS 1220 (N-[9-Chloro-2-(2-furanyl)[1,2,4]-triazolo [1,5-c]quinazolin-5-yl]benzene acetamide). Finally, ischemia-induced injury and functional loss to the optic nerve was attenuated by blocking A3 receptors. Together, these results indicate that adenosine may trigger oligodendrocyte death via activation of A3 receptors and suggest that this mechanism contributes to optic nerve and white matter ischemic damage.

  7. A Cistanches Herba Fraction/β-Sitosterol Causes a Redox-Sensitive Induction of Mitochondrial Uncoupling and Activation of Adenosine Monophosphate-Dependent Protein Kinase/Peroxisome Proliferator-Activated Receptor γ Coactivator-1 in C2C12 Myotubes: A Possible Mechanism Underlying the Weight Reduction Effect

    Hoi Shan Wong


    Full Text Available Previous studies have demonstrated that HCF1, a semipurified fraction of Cistanches Herba, causes weight reduction in normal diet- and high fat diet-fed mice. The weight reduction was associated with the induction of mitochondrial uncoupling and changes in metabolic enzyme activities in mouse skeletal muscle. To further investigate the biochemical mechanism underlying the HCF1-induced weight reduction, the effect of HCF1 and its active component, β-sitosterol (BSS, on C2C12 myotubes was examined. Incubation with HCF1/BSS caused a transient increase in mitochondrial membrane potential (MMP, possibly by fluidizing the mitochondrial inner membrane. The increase in MMP was paralleled to an increase in mitochondrial reactive oxygen species (ROS production. Mitochondrial ROS, in turn, triggered a redox-sensitive induction of mitochondrial uncoupling by uncoupling protein 3 (UCP3. Biochemical analysis indicated that HCF1 was capable of activating an adenosine monophosphate-dependent protein kinase/peroxisome proliferator-activated receptor γ coactivator-1 pathway and thereby increased the expression of cytochrome c oxidase and UCP3. Animal studies using mitochondrial recoupler also confirmed the role of mitochondrial uncoupling in the HCF1-induced weight reduction. In conclusion, a HCF1/BSS causes the redox-sensitive induction of mitochondrial uncoupling and activation of AMPK/PGC-1 in C2C12 myotubes, with resultant reductions in body weight and adiposity by increased energy consumption.

  8. Purification and characteristics of functional properties of soluble nucleoside triphosphatase (apyrase) from bovine brain.

    Sivuk, V F; Rusina, I M; Makarchikov, A F


    Soluble NTPase, differing in its properties from known proteins exhibiting NTPase activity, was purified from bovine brain to homogeneity. The enzyme has pH optimum at 7.5 and shows absolute dependence on bivalent cations and broad substrate specificity towards nucleoside-5 -tri- and -diphosphates, characteristics of apyrases. The NTPase follows Michaelis-Menten kinetics in the range of investigated substrate concentrations, the apparent K(m) values for UTP, ITP, GTP, CTP, CDP, and ATP being 86, 25, 41, 150, 500, and 260 microM, respectively. According to gel-filtration and SDS-PAGE data, the molecular mass of the enzyme is 60 kD. The NTPase is localized in the cytosol fraction and expressed in different bovine organs and tissues. Total NTPase activity of extracts of bovine organs and tissues decreases in the following order: liver > heart > skeletal muscle > lung > brain > spleen > kidney ~ small intestine. The enzyme activity can be regulated by acetyl-CoA, alpha-ketoglutarate, and fructose-1,6-diphosphate acting as activators in physiological concentrations, whereas propionate exhibits an inhibitory effect.

  9. Downregulation of adenosine and P2X receptor-mediated cardiovascular responses in heart failure rats

    Zhao, Xin; Sun, X Y; Erlinge, D;


    Neurohormonal changes in congestive heart failure (CHF) include an enhanced peripheral sympathetic nerve activity which results in increased release of noradrenaline, neuropeptide Y and ATP. To examine if such changes in CHF would modulate peripheral pre- and postsynaptic receptors of ATP and its...... effects mediated by the endothelial P2Y receptors are unaffected in CHF. Moreover, the adenosine-mediated inhibitory effects on heart rate and blood pressure were also attenuated in the CHF rats. The most important changes in adenosine and P2-receptor function induced by ischaemic CHF were the reduced...... pressor effect mediated by the P2X receptor and the increased heart rate due to an attenuated inhibitory effect of adenosine....

  10. Regulation of adenosine triphosphate-sensitive potassium channels suppresses the toxic effects of amyloid-beta peptide (25-35)

    Min Kong; Maowen Ba; Hui Liang; Peng Shao; Tianxia Yu; Ying Wang


    In this study, we treated PC12 cells with 0-20 μM amyloid-β peptide (25-35) for 24 hours to induce cytotoxicity, and found that 5-20 μM amyloid-β peptide (25-35) decreased PC12 cell viability, but adenosine triphosphate-sensitive potassium channel activator diazoxide suppressed the decrease reactive oxygen species levels. These protective effects were reversed by the selective mitochondrial adenosine triphosphate-sensitive potassium channel blocker 5-hydroxydecanoate. An inducible nitric oxide synthase inhibitor, Nω-nitro-L-arginine, also protected PC12 cells from intracellular reactive oxygen species levels. However, the H2O2-degrading enzyme catalase could that the increases in both mitochondrial membrane potential and reactive oxygen species levels adenosine triphosphate-sensitive potassium channels and nitric oxide. Regulation of adenosine triphosphate-sensitive potassium channels suppresses PC12 cell cytotoxicity induced by amyloid-β

  11. Adenosine receptor antagonists alter the stability of human epileptic GABAA receptors

    Roseti, Cristina; Martinello, Katiuscia; Fucile, Sergio; Piccari, Vanessa; Mascia, Addolorata; Di Gennaro, Giancarlo; Quarato, Pier Paolo; Manfredi, Mario; Esposito, Vincenzo; Cantore, Gianpaolo; Arcella, Antonella; Simonato, Michele; Fredholm, Bertil B.; Limatola, Cristina; Miledi, Ricardo; Eusebi, Fabrizio


    We examined how the endogenous anticonvulsant adenosine might influence γ-aminobutyric acid type A (GABAA) receptor stability and which adenosine receptors (ARs) were involved. Upon repetitive activation (GABA 500 μM), GABAA receptors, microtransplanted into Xenopus oocytes from neurosurgically resected epileptic human nervous tissues, exhibited an obvious GABAA-current (IGABA) run-down, which was consistently and significantly reduced by treatment with the nonselective adenosine receptor antagonist CGS15943 (100 nM) or with adenosine deaminase (ADA) (1 units/ml), that inactivates adenosine. It was also found that selective antagonists of A2B (MRS1706, 10 nM) or A3 (MRS1334, 30 nM) receptors reduced IGABA run-down, whereas treatment with the specific A1 receptor antagonist DPCPX (10 nM) was ineffective. The selective A2A receptor antagonist SCH58261 (10 nM) reduced or potentiated IGABA run-down in ≈40% and ≈20% of tested oocytes, respectively. The ADA-resistant, AR agonist 2-chloroadenosine (2-CA) (10 μM) potentiated IGABA run-down but only in ≈20% of tested oocytes. CGS15943 administration again decreased IGABA run-down in patch-clamped neurons from either human or rat neocortex slices. IGABA run-down in pyramidal neurons was equivalent in A1 receptor-deficient and wt neurons but much larger in neurons from A2A receptor-deficient mice, indicating that, in mouse cortex, GABAA-receptor stability is tonically influenced by A2A but not by A1 receptors. IGABA run-down from wt mice was not affected by 2-CA, suggesting maximal ARs activity by endogenous adenosine. Our findings strongly suggest that cortical A2–A3 receptors alter the stability of GABAA receptors, which could offer therapeutic opportunities. PMID:18809912

  12. Day-night variations of adenosine and its metabolizing enzymes in the brain cortex of the rat--possible physiological significance for the energetic homeostasis and the sleep-wake cycle.

    Chagoya de Sánchez, V; Hernández Múñoz, R; Suárez, J; Vidrio, S; Yáñez, L; Díaz Múñoz, M


    The role of adenosine as a metabolic regulator of physiological processes in the brain was studied by measuring its concentrations and the activity of adenosine-metabolizing enzymes: 5'-nucleotidase, S-adenosylhomocysteine hydrolase, adenosine deaminase and adenosine kinase in the cerebral cortex of the rat. Other purine compounds, such as, inosine, hypoxanthine and adenine nucleotides were also studied. The purines' pattern was bimodal with high levels of adenosine, inosine and hypoxanthine during the light period reaching their peak at 12.00 h, 08.00 h and 16.00 h, respectively, and small increments during the night between 02.00 h and 04.00 h. The enzymatic activities showed, in general, an unimodal profile with low activity during the day and high activities at night. The adenine nucleotide profile showed a significant diminution between 12.00 h and 24.00 h. The high adenosine level during the day might be due to a diminution of adenine nucleotide and to the low activity of adenosine-metabolizing enzymes, suggesting an accumulation of the nucleoside. The night increase, although of less magnitude, is simultaneous to high activity of adenosine-metabolizing enzymes and could be due to an increased formation of the nucleoside. The present data and the findings from other authors strongly suggest that adenosine in the brain cortex of the rat can participate at least in two physiological processes: regulation of the sleep-wake cycle and replenishment of the adenine nucleotide pool.

  13. Adenosine elicits an eNOS-independent reduction in arterial blood pressure in conscious mice that involves adenosine A(2A) receptors

    Andersen, Henrik; Jaff, Mohammad G; Høgh, Ditte;


    Aims:  Adenosine plays an important role in the regulation of heart rate and vascular reactivity. However, the mechanisms underlying the acute effect of adenosine on arterial blood pressure in conscious mice are unclear. Therefore, the present study investigated the effect of the nucleoside on mean...... arterial blood pressure (MAP) and heart rate (HR) in conscious mice. Methods:  Chronic indwelling catheters were placed in C57Bl/6J (WT) and endothelial nitric oxide synthase knock-out (eNOS(-/-) ) mice for continuous measurements of MAP and HR. Using PCR and myograph analysis involment of adenosine...... receptors was investigated in human and mouse renal blood vessels Results:  Bolus infusion of 0.5 mg/kg adenosine elicited significant transient decreases in MAP (99.3±2.3 to 70.4±4.5 mmHg) and HR (603.2±18.3 to 364.3±49.2 min(-1) ) which were inhibited by the A(2A) receptor antagonist ZM 241385. Activation...

  14. Adenosine as a signaling molecule in the retina: biochemical and developmental aspects



    Full Text Available The nucleoside adenosine plays an important role as a neurotransmitter or neuromodulator in the central nervous system, including the retina. In the present paper we review compelling evidence showing that adenosine is a signaling molecule in the developing retina. In the chick retina, adenosine transporters are present since early stages of development before the appearance of adenosine A1 receptors modulating dopamine-dependent adenylate cyclase activity or A2 receptors that directly activate the enzyme. Experiments using retinal cell cultures revealed that adenosine is taken up by specific cell populations that when stimulated by depolarization or neurotransmitters such as dopamine or glutamate, release the nucleoside through calcium-dependent transporter-mediated mechanisms. The presence of adenosine in the extracellular medium and the long-term activation of adenosine receptors is able to regulate the survival of retinal neurons and blocks glutamate excitoxicity. Thus, adenosine besides working as a neurotransmitter or neuromodulator in the mature retina, is considered as an important signaling molecule during retinal development having important functions such as regulation of neuronal survival and differentiation.O nucleosídeo adenosina apresenta um importante papel como neurotransmissor ou neuromodulador no sistema nervoso central, inclusive na retina. Neste artigo apresentamos uma revisão das evidências que mostram que a adenosina é uma molécula sinalizadora na retina em desenvolvimento. Na retina de pinto, transportadores de adenosina estão presentes desde estágios precoces do desenvolvimento, antes do aparecimento dos receptores A1 que modulam a atividade adenilato ciclase dependente de dopamina ou dos receptores A2 que ativam diretamente a enzima. Experimentos usando culturas de células de retina revelaram que a adenosina é captada por populações celulares específicas que, quando estimuladas por despolarização ou por

  15. A3 Adenosine Receptors Modulate Hypoxia-inducible Factor-1a Expression in Human A375 Melanoma Cells

    Stefania Merighi


    Full Text Available Hypoxia-inducible factor-1 (HIF-1 is a key regulator of genes crucial to many aspects of cancer biology. The purine nucleoside, adenosine, accumulates within many tissues under hypoxic conditions, including that of tumors. Because the levels of both HIF-1 and adenosine are elevated within the hypoxic environment of solid tumors, we investigated whether adenosine may regulate HIF-1. Here we show that, under hypoxic conditions (< 2% 02, adenosine upregulates HIF-1α protein expression in a dose-dependent and timedependent manner, exclusively through the A3 receptor subtype. The response to adenosine was generated at the cell surface because the inhibition of A3 receptor expression, by using small interfering RNA, abolished nucleoside effects. A3 receptor stimulation in hypoxia also increases angiopoietin-2 (Ang-2 protein accumulation through the induction of HIF-1α. In particular, we found that A3 receptor stimulation activates p44/p42 and p38 mitogen-activated protein kinases, which are required for A3-induced increase of HIF-1a and Ang-2. Collectively, these results suggest a cooperation between hypoxic and adenosine signals that ultimately may lead to the increase in HIF-1-mediated effects in cancer cells.

  16. A non-invasive method of measuring concentrations of rubidium in rat skeletal muscle in vivo by 87Rb nuclear magnetic resonance spectroscopy: implications for the measurement of cation transport activity in vivo.

    Syme, P D; Dixon, R M; Allis, J L; Aronson, J K; Grahame-Smith, D G; Radda, G K


    1. We have used n.m.r. spectroscopy to measure rubidium concentrations in the skeletal muscle of live intact rats. Using a 1.9 T superconducting magnet and an ear-phone coil tuned to both protons (1H) and rubidium (87Rb), it was possible to make measurements of both tissue rubidium content and water content, and from these measurements to obtain the rubidium concentration. 2. The n.m.r. estimate of rubidium concentration in muscle in vivo was found to be a constant 31% (SEM 4%) of that estimated by flame atomic absorption spectroscopy in an extract of excised muscle. This is close to the predicted theoretical n.m.r. visibility of 33%. The visibility was constant for muscle rubidium concentrations ranging between 10 and 34 mmol/l. 3. Rubidium concentration measurement by this method is unaffected by variations in sample geometry, sample volume, tissue conductivity, coil tuning and amplifier gain. 4. By using this method to measure changes in tissue rubidium concentration with time in the same animal, it should now be possible to assess the activity of ion transport systems, such as sodium- and potassium-activated adenosine triphosphatase in vivo, by measuring the rates of change of tissue rubidium concentrations during the administration of rubidium salts. 5. This method could also be used to measure the absolute concentration of any n.m.r.-visible nucleus and could be applied to man.

  17. Cyclic adenosine 3'-5'-monophosphate (cAMP) exerts proliferative and anti-proliferative effects in pituitary cells of different types by activating both cAMP-dependent protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac).

    Vitali, E; Peverelli, E; Giardino, E; Locatelli, M; Lasio, G B; Beck-Peccoz, P; Spada, A; Lania, A G; Mantovani, G


    In the pituitary the activation of cyclic adenosine 3'-5'-monophosphate (cAMP) dependent pathways generates proliferative signals in somatotrophs, whereas in pituitary cells of other lineages its effect remains uncertain. Moreover, the specific role of the two main cAMP effectors, protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac), has not been defined. Aim of this study was to investigate the effect of cAMP on pituitary adenomatous cells proliferation and to identify PKA and Epac differential involvement. We found that cAMP increased DNA synthesis and cyclin D1 expression in somatotropinomas, whereas it reduced both parameters in prolactinomas and nonfunctioning adenomas, these effects being replicated in corresponding cell lines. Moreover, the divergent cAMP effects were mimicked by Epac and PKA analogs, which activated Rap1 and CREB, respectively. In conclusion, we demonstrated that cAMP exerted opposite effects on different pituitary cell types proliferation, these effects being mediated by both Epac and PKA.

  18. The effect of cannabidiol on ischemia/reperfusion-induced ventricular arrhythmias: the role of adenosine A1 receptors.

    Gonca, Ersöz; Darıcı, Faruk


    Cannabidiol (CBD) is a nonpsychoactive phytocannabinoid with anti-inflammatory activity mediated by enhancing adenosine signaling. As the adenosine A1 receptor activation confers protection against ischemia/reperfusion (I/R)-induced ventricular arrhythmias, we hypothesized that CBD may have antiarrhythmic effect through the activation of adenosine A1 receptor. Cannabidiol has recently been shown to suppress ischemia-induced ventricular arrhythmias. We aimed to research the effect of CBD on the incidence and the duration of I/R-induced ventricular arrhythmias and to investigate the role of adenosine A1 receptor activation in the possible antiarrhythmic effect of CBD. Myocardial ischemia and reperfusion was induced in anesthetized male rats by ligating the left anterior descending coronary artery for 6 minutes and by loosening the bond at the coronary artery, respectively. Cannabidiol alone was given in a dose of 50 µg/kg, 10 minutes prior to coronary artery occlusion and coadministrated with adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) in a dose of 100 µg/kg, 15 minutes prior to coronary artery occlusion to investigate whether the antiarrhythmic effect of CBD is modified by the activation of adenosine A1 receptors. The experimental groups were as follows: (1) vehicle control (n = 10), (2) CBD (n = 9), (3) DPCPX (n = 7), and (4) CBD + DPCPX group (n = 7). Cannabidiol treatment significantly decreased the incidence and the duration of ventricular tachycardia, total length of arrhythmias, and the arrhythmia scores compared to control during the reperfusion period. The DPCPX treatment alone did not affect the incidence and the duration of any type of arrhythmias. However, DPCPX aborted the antiarrhythmic effect of CBD when it was combined with it. The present results demonstrated that CBD has an antiarrhythmic effect against I/R-induced arrhythmias, and the antiarrhythmic effect of CBD may be mediated through the activation of adenosine

  19. Magnetically assisted fluorescence ratiometric assays for adenosine deaminase using water-soluble conjusated polymers

    HE Fang; YU MingHui; WANG Shu


    A magnetically assisted fluorescence ratiometric technique has been developed for adenosine deami-nase assays with high sensitivity using water-soluble cationic conjugated polymers (CCPs).The assay contains three elements:a biotin-labeled aptamer of adenosine (biotin-aptamer),a signaling probe single-stranded DNA-tagged fiuorescein at terminus (ssDNA-FI) and a CCP.The specific binding of adenosine to biotin-aptamer makes biotin-aptamer and ssDNA-FI unhybridized,and the ssDNA-FI is washed out after streptavidin-coated magnetic beads are added and separated from the assay solution under magnetic field.In this case,after the addition of CCP to the magnetic beads solution,the fluo-rescence resonance energy transfer (FRET) from CCP to fluorescein is inefficient.Upon adding adenosine deaminase,the adenosine is converted into inosine,and the biotin-aptamer is hybridized with ssDNA-FI to form doubled stranded DNA (biotin-dsDNA-FI).The ssONA-FI is attached to the mag-netic beads at the separation step,and the addition of CCP to the magnetic beads solution leads to efficient FRET from CCP to fluorescein.Thus the adenosine deaminase activity can be monitored by fluorescence spectra in view of the intensity decrease of CCP emission or the increase of fluorescein emission in aqueous solutions.The assay integrates surface-functionalized magnetic particles with significant amplification of detection signal of water-soluble cationic conjugated polymers.

  20. Aminopyrimidine derivatives as adenosine antagonists / Janke Kleynhans

    Kleynhans, Janke


    Aims of this project - The aim of this study was to design and synthesise novel 2-aminopyrimidine derivatives as potential adenosine A1 and A2A receptor antagonists. Background and rationale - Parkinson’s disease is the second most common neurodegenerative disorder (after Alzheimer’s disease) and is characterised by the selective death of the dopaminergic neurons of the nigro-striatal pathway. Distinctive motor symptoms include bradykinesia, muscle rigidity and tremor, while non-m...

  1. Production of adenosine from extracellular ATP at the striatal cholinergic synapse.

    James, S; Richardson, P J


    The components of the ectonucleotidase pathway at the immunoaffinity-purified striatal cholinergic synapse have been studied. The ecto-ATPase (EC had a Km of 131 microM, whereas the ecto-ADPase (EC had a Km of 58 microM, was Ca(2+)-dependent, and was inhibited by the ATP analogue 5'-adenylylimidodiphosphate (AMPPNP). The ecto-5'-nucleotidase (EC had a Km of 21 microM, was inhibited by AMPPNP and alpha,beta-methylene ADP, and by a specific antiserum. The Vmax values of the ATPase, ADPase, and 5'-nucleotidase enzymes present at this synapse were in a ratio of 30:14:1. Very little ecto-adenylate kinase activity was detected on these purified synapses. The intraterminal 5'-nucleotidase enzyme, which amounted to 40% of the total 5'-nucleotidase activity, was inhibited by AMPPNP, alpha,beta-methylene ADP, and the antiserum, and also had the same kinetic properties as the ectoenzyme. The time course of ATP degradation to adenosine outside the nerve terminals showed a delay, followed by a period of sustained adenosine production. The delay in adenosine production was proportional to the initial ATP concentration, was a consequence of feedforward inhibition of the ADPase and 5'-nucleotidase, and was inversely proportional to the ecto-5'-nucleotidase activity. The function and characteristics of this pathway and the central role of 5'-nucleotidase in the regulation of extraterminal adenosine concentrations are discussed.

  2. Presynaptic inhibition by kainate receptors converges mechanistically with presynaptic inhibition by adenosine and GABAB receptors.

    Partovi, Dara; Frerking, Matthew


    Kainate receptors are widely reported to regulate the release of neurotransmitter in the CNS, but the mechanisms involved remain controversial. Previous studies have found that the kainate receptor agonist ATPA, which selectively activates Glu(K5)-containing kainate receptors, depresses glutamate release at Schaffer-collateral synapses in the hippocampus. In the present study, we provide pharmacological evidence that this depressant effect is mediated by Glu(K5)-containing heteromers, but is distinct from a similar depressant effect engaged by the kainate receptor agonist domoate. The depressant effect of ATPA is insensitive to antagonists for GABA(A), GABA(B), and adenosine receptors, and is also unaffected by lowering the release probability by reducing extracellular calcium. However, the effect of ATPA is partly occluded by prior activation of GABA(B) receptors and completely occluded by prior activation of adenosine receptors, suggesting a mechanistic convergence of heteromeric Glu(K5) kainate receptor signaling with GABA(B) receptors and adenosine receptors. The effects of domoate are partially occluded by both adenosine and GABA(B) receptor agonists, indicating at least a partial convergence of Glu(K5)-lacking kainate receptor signaling with these other pathways. The depressant effect of ATPA is not blocked by inhibition of serine/threonine protein kinases. These results suggest that ATPA and domoate inhibit glutamate release through mechanisms that converge with those of classical metabotropic receptor agonists, although they do so through different receptors.

  3. Ischemic preconditioning protects post-ischemic renal function in anesthetized dogs: role of adenosine and adenine nucleotides

    Fan-zhu LI; Shoji KIMURA; Akira NISHIYAMA; Matlubur RAHMAN; Guo-xing ZHANG; Youichi ABE


    Aim: To investigate the effects of renal ischemic preconditioning (IPC) on both renal hemodynamics and the renal interstitial concentrations of adenosine and adenine nucleotides induced by ischemia-reperfusion injury.Methods: Renal hemodynamics responses to ischemia-reperfusion injury in mongrel dog models were determined with or without multiple brief renal ischemic preconditioning treatments, as well as the adenosine A1 receptor antagonist (KW-3902),respectively.The renal interstitial concentrations of adenosine and adenine nucleotides in response to ischemia-reperfusion injury, either following 1-3 cycles of IPC or not, were measured simultaneously using microdialysis sampling technology.Results: One 10-min IPC, adenosine A1 receptor antagonist (KW3902) also shortened the recovery time of renal blood flow (RBF) and urine flow (UF), as well as mean blood pressure (BP).Advanced renal IPC attenuated the increment of adenosine and adenine nucleotides, as well as recovery time during the 60-min reperfusion which followed the 60-min renal ischemia.All of these recovery times were dependent on the cycles of 10-min IPC.The renal interstitial concentrations of adenosine and adenine nucleotides increased and decreased during renal ischemia and reperfusion, respectively.Conclusion: A significant relativity in dog models exists between the cycles of 10-min renal IPC and the recovery time of BP, UF, and RBF during the 60-min renal reperfusion following 60-min renal ischemia, respectively.Renal IPC can protect against ischemiareperfusion injury and the predominant effect of endogenous adenosine induced by prolonged renal ischemia; renal adenosine A1 receptor activation during the renal ischemia-reperfusion injury is detrimental to renal function.

  4. Joint detection of adenosine deaminase activity and IFN-γ in the application of the identification diagnosis on tuberculous pleurisy%腺苷脱氨酶和干扰素-γ联合检测在结核性胸膜炎鉴别诊断中的应用

    刘莉敏; 李玉磊; 谢艳丽; 王以炳; 李琪


    目的 研究腺苷脱氨酶(ADA)和干扰素-γ联合检测对结核性胸膜炎诊断价值.方法 检测60例呼吸内科患者胸腔积液中ADA活性和干扰素-γ水平.并绘制ADA活性和干扰素-γ水平两者单项指标的ROC曲线.结果结核性胸液组的ADA和干扰素-γ高于恶性胸液组(P<0.001);ROC曲线显示两项指标均具有较高的敏感性和特异性,ADA的诊断价值高于IFN-γ;ADA和干扰素-γ联合检测优于单项.结论 ADA检测可作为结核性胸膜炎的重要指标,ADA和IFN-γ联合检测可以提高诊断试验的效率.%Objective To research the identification and diagnosis value of the joint detection of adenosine deaminase activity and IFN-γ on tuberculous pleurisy. Methods ADA activity and IFN-γ were detected in 60 respiration medicine patients' pleural effusion and the ROC curve of single index between ADA activity and IFN--γ was draw. Results The pleural fluid levels of adenosine deaminase and IFN-γ were significantly higher in the tuberculous pleufitis group than that in the malignant pleuritis group ( P < 0. 001 ). ROC curve showed that two indexes both had the character of sensibility and specificity. The diagnosis value of ADA was higher than that of IFN-γ and joint detection was better than the single index. Conclusion Detection of ADA in pleural fluid can be regarded as one important index to diagnose tuberculous pleurisy. Joint detection of adenosine deaminase activity and interleron-γ in tuberculous pleural effusions can improve the efficiency of diagnostic tests.

  5. A2BR adenosine receptor modulates sweet taste in circumvallate taste buds.

    Shinji Kataoka

    Full Text Available In response to taste stimulation, taste buds release ATP, which activates ionotropic ATP receptors (P2X2/P2X3 on taste nerves as well as metabotropic (P2Y purinergic receptors on taste bud cells. The action of the extracellular ATP is terminated by ectonucleotidases, ultimately generating adenosine, which itself can activate one or more G-protein coupled adenosine receptors: A1, A2A, A2B, and A3. Here we investigated the expression of adenosine receptors in mouse taste buds at both the nucleotide and protein expression levels. Of the adenosine receptors, only A2B receptor (A2BR is expressed specifically in taste epithelia. Further, A2BR is expressed abundantly only in a subset of taste bud cells of posterior (circumvallate, foliate, but not anterior (fungiform, palate taste fields in mice. Analysis of double-labeled tissue indicates that A2BR occurs on Type II taste bud cells that also express Gα14, which is present only in sweet-sensitive taste cells of the foliate and circumvallate papillae. Glossopharyngeal nerve recordings from A2BR knockout mice show significantly reduced responses to both sucrose and synthetic sweeteners, but normal responses to tastants representing other qualities. Thus, our study identified a novel regulator of sweet taste, the A2BR, which functions to potentiate sweet responses in posterior lingual taste fields.

  6. Adenosine A2A Receptors Modulate Acute Injury and Neuroinflammation in Brain Ischemia

    Felicita Pedata


    Full Text Available The extracellular concentration of adenosine in the brain increases dramatically during ischemia. Adenosine A2A receptor is expressed in neurons and glial cells and in inflammatory cells (lymphocytes and granulocytes. Recently, adenosine A2A receptor emerged as a potential therapeutic attractive target in ischemia. Ischemia is a multifactorial pathology characterized by different events evolving in the time. After ischemia the early massive increase of extracellular glutamate is followed by activation of resident immune cells, that is, microglia, and production or activation of inflammation mediators. Proinflammatory cytokines, which upregulate cell adhesion molecules, exert an important role in promoting recruitment of leukocytes that in turn promote expansion of the inflammatory response in ischemic tissue. Protracted neuroinflammation is now recognized as the predominant mechanism of secondary brain injury progression. A2A receptors present on central cells and on blood cells account for important effects depending on the time-related evolution of the pathological condition. Evidence suggests that A2A receptor antagonists provide early protection via centrally mediated control of excessive excitotoxicity, while A2A receptor agonists provide protracted protection by controlling massive blood cell infiltration in the hours and days after ischemia. Focus on inflammatory responses provides for adenosine A2A receptor agonists a wide therapeutic time-window of hours and even days after stroke.

  7. Inhibition of adenosine deaminase attenuates endotoxin-induced release of cytokines in vivo in rats.

    Tofovic, S P; Zacharia, L; Carcillo, J A; Jackson, E K


    The purpose of this study was to investigate in vivo the effects of modulating the adenosine system on endotoxin-induced release of cytokines and changes in heart performance and neurohumoral status in early, profound endotoxemia in rats. Time/pressure variables of heart performance and blood pressure were recorded continuously, and plasma levels of tumor necrosis factor alpha (TNFalpha), interleukin 1-beta (IL-1beta), plasma renin activity (PRA), and catecholamines were determined before and 90 min after administration of endotoxin (30 mg/kg of lipopolysaccharide, i.v.). Erythro-9[2-hydroxyl-3-nonyl] adenine (EHNA; an adenosine deaminase inhibitor) had no effects on measured time-pressure variables of heart performance under baseline conditions and during endotoxemia, yet significantly attenuated endotoxin-induced release of cytokines and PRA. Pretreatment with the non-selective adenosine receptor antagonist DPSPX not only prevented the effects of EHNA but also increased the basal release of cytokines and augmented PRA. At baseline, caffeine (a non-selective adenosine receptor antagonist) increased HR, +dP/dtmax, heart rate x ventricular pressure product (HR x VPSP) and +dP/dtmax normalized by pressure (+dP/dtmax/VPSP), and these changes persisted during endotoxemia. Caffeine attenuated endotoxin-induced release of cytokines and augmented endotoxin-induced increases in plasma catecholamines and PRA. Pretreatment with propranolol abolished the effects of caffeine on heart performance and neurohumoral activation during the early phase of endotoxemia. 6N-cyclopentyladenosine (CPA; selective A1 adenosine receptor agonist) induced bradicardia and negative inotropic effects, reduced work load (i.e., decreased HR, VPSP, +dP/dtmax, +dP/dtmax/VPSP and HR x VPSP) and inhibited endotoxin-induced tachycardia and renin release. CGS 21680 (selective A2A adenosine receptor agonist) decreased blood pressure under basal condition but did not potentiate decreases in blood pressure

  8. Twenty-four-hour changes of S-adenosylmethionine, S-adenosylhomocysteine adenosine and their metabolizing enzymes in rat liver; possible physiological significance in phospholipid methylation.

    Chagoya de Sánchez, V; Hernández-Muñoz, R; Sánchez, L; Vidrio, S; Yáñez, L; Suárez, J


    1. The metabolic control of adenosine concentration in the rat liver through the 24-hr cycle is related to the activity of adenosine-metabolizing enzymes [5'-nucleotidase (5'N), adenosine deaminase (A.D.), adenosine kinase (A.K.) and S-adenosylhomocysteine hydrolase (SAH-H)]. 2. Two peaks of adenosine were observed, one at 12:00 hr caused by high activity of 5'N and SAH-H, and the other at 02:00 hr, caused by a decrease in purine catabolism and purine utilization, low activity of SAH-H and de novo purine formation. 3. The similarity of the adenosine and S-adenosylmethionine (SAM) profiles through the 24-hr cycle suggests a role of adenosine in transmethylation reactions, because, during the night (02:00 hr), the metabolic conditions favor the formation and accumulation of S-adenosylhomocysteine (SAH), with consequent inhibition of transmethylation reactions. 4. In the 24-hr variation of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), the lowest ratio of PC/PE was observed at 24:00-02:00 hr when SAH concentration is high, whereas the highest PC/PE ratio occurs at the same time as one of the SAM/SAH ratio maxima.

  9. Adenosine Receptors Differentially Regulate the Expression of Regulators of G-Protein Signalling (RGS 2, 3 and 4 in Astrocyte-Like Cells.

    Till Nicolas Eusemann

    Full Text Available The "regulators of g-protein signalling" (RGS comprise a large family of proteins that limit by virtue of their GTPase accelerating protein domain the signal transduction of G-protein coupled receptors. RGS proteins have been implicated in various neuropsychiatric diseases such as schizophrenia, drug abuse, depression and anxiety and aggressive behaviour. Since conditions associated with a large increase of adenosine in the brain such as seizures or ischemia were reported to modify the expression of some RGS proteins we hypothesized that adenosine might regulate RGS expression in neural cells. We measured the expression of RGS-2,-3, and -4 in both transformed glia cells (human U373 MG astrocytoma cells and in primary rat astrocyte cultures stimulated with adenosine agonists. Expression of RGS-2 mRNA as well as RGS2 protein was increased up to 30-fold by adenosine agonists in astrocytes. The order of potency of agonists and the blockade by the adenosine A2B-antagonist MRS1706 indicated that this effect was largely mediated by adenosine A2B receptors. However, a smaller effect was observed due to activation of adenosine A2A receptors. In astrocytoma cells adenosine agonists elicited an increase in RGS-2 expression solely mediated by A2B receptors. Expression of RGS-3 was inhibited by adenosine agonists in both astrocytoma cells and astrocytes. However while this effect was mediated by A2B receptors in astrocytoma cells it was mediated by A2A receptors in astrocytes as assessed by the order of potency of agonists and selective blockade by the specific antagonists MRS1706 and ZM241385 respectively. RGS-4 expression was inhibited in astrocytoma cells but enhanced in astrocytes by adenosine agonists.

  10. Primary adenosine monophosphate (AMP) deaminase deficiency in a hypotonic infant.

    Castro-Gago, Manuel; Gómez-Lado, Carmen; Pérez-Gay, Laura; Eirís-Puñal, Jesús; Martínez, Elena Pintos; García-Consuegra, Inés; Martín, Miguel Angel


    The spectrum of the adenosine monophosphate (AMP) deaminase deficiency ranges from asymptomatic carriers to patients who manifest exercise-induced muscle pain, occasionally rhabdomyolysis, and idiopathic hyperCKemia. However, previous to the introduction of molecular techniques, rare cases with congenital weakness and hypotonia have also been reported. We report a 6-month-old girl with the association of congenital muscle weakness and hypotonia, muscle deficiency of adenosine monophosphate deaminase, and the homozygous C to T mutation at nucleotide 34 of the adenosine monophosphate deaminase-1 gene. This observation indicates the possible existence of a primary adenosine monophosphate deaminase deficiency manifested by congenital muscle weakness and hypotonia.

  11. cAMP-independent dilation of coronary arterioles to adenosine : role of nitric oxide, G proteins, and K(ATP) channels.

    Hein, T W; Kuo, L


    Adenosine is known to play an important role in the regulation of coronary blood flow during metabolic stress. However, there is sparse information on the mechanism of adenosine-induced dilation at the microcirculatory levels. In the present study, we examined the role of endothelial nitric oxide (NO), G proteins, cyclic nucleotides, and potassium channels in coronary arteriolar dilation to adenosine. Pig subepicardial coronary arterioles (50 to 100 microm in diameter) were isolated, cannulated, and pressurized to 60 cm H(2)O without flow for in vitro study. The arterioles developed basal tone and dilated dose dependently to adenosine. Disruption of endothelium, blocking of endothelial ATP-sensitive potassium (K(ATP)) channels by glibenclamide, and inhibition of NO synthase by N(G)-nitro-L-arginine methyl ester and of soluble guanylyl cyclase by 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one produced identical attenuation of vasodilation to adenosine. Combined administration of these inhibitors did not further attenuate the vasodilatory response. Production of NO from coronary arterioles was significantly increased by adenosine. Pertussis toxin, but not cholera toxin, significantly inhibited vasodilation to adenosine, and this inhibitory effect was only evident in vessels with an intact endothelium. Tetraethylammonium, glibenclamide, and a high concentration of extraluminal KCl abolished vasodilation of denuded vessels to adenosine; however, inhibition of calcium-activated potassium channels by iberiotoxin had no effect on this dilation. Rp-8-Br-cAMPS, a cAMP antagonist, inhibited vasodilation to cAMP analog 8-Br-cAMP but failed to block adenosine-induced dilation. Furthermore, vasodilations to 8-Br-cAMP and sodium nitroprusside were not inhibited by glibenclamide, indicating that cAMP- and cGMP-induced dilations are not mediated by the activation of K(ATP) channels. These results suggest that adenosine activates both endothelial and smooth muscle pathways to exert

  12. Central or peripheral delivery of an adenosine A1 receptor agonist improves mechanical allodynia in a mouse model of painful diabetic neuropathy.

    Katz, N K; Ryals, J M; Wright, D E


    Diabetic peripheral neuropathy is a common complication of diabetes mellitus, and a significant proportion of individuals suffer debilitating pain that significantly affects their quality of life. Unfortunately, symptomatic treatment options have limited efficacy, and often carry significant risk of systemic adverse effects. Activation of the adenosine A1 receptor (A1R) by the analgesic small molecule adenosine has been shown to have antinociceptive benefits in models of inflammatory and neuropathic pain. The current study used a mouse model of painful diabetic neuropathy to determine the effect of diabetes on endogenous adenosine production, and if central or peripheral delivery of adenosine receptor agonists could alleviate signs of mechanical allodynia in diabetic mice. Diabetes was induced using streptozocin in male A/J mice. Mechanical withdrawal thresholds were measured weekly to characterize neuropathy phenotype. Hydrolysis of AMP into adenosine by ectonucleotidases was determined in the dorsal root ganglia (DRG) and spinal cord at 8 weeks post-induction of diabetes. AMP, adenosine and the specific A1R agonist, N(6)-cyclopentyladenosine (CPA), were administered both centrally (intrathecal) and peripherally (intraplantar) to determine the effect of activation of adenosine receptors on mechanical allodynia in diabetic mice. Eight weeks post-induction, diabetic mice displayed significantly decreased hydrolysis of extracellular AMP in the DRG; at this same time, diabetic mice displayed significantly decreased mechanical withdrawal thresholds compared to nondiabetic controls. Central delivery AMP, adenosine and CPA significantly improved mechanical withdrawal thresholds in diabetic mice. Surprisingly, peripheral delivery of CPA also improved mechanical allodynia in diabetic mice. This study provides new evidence that diabetes significantly affects endogenous AMP hydrolysis, suggesting that altered adenosine production could contribute to the development of

  13. Effects of AMP579 and adenosine on L-type Ca2+ current in isolated rat ventricular myocytes

    Xiong WANG; Bo-wei WU; Dong-mei WU


    Aim: To compare the effects of AMP579 and adenosine on L-type Ca2+ current (ICa- L) in rat ventricular myocytes and explore the mechanism by which AMP579 acts on ICa-L. Methods: ICa-L was recorded by patch-clamp technique in whole-cell configuration. Results: Adenosine (10 nmol/L to 50 μmol/L) showed no effect on basal ICa- L, but it inhibited the ICa-L induced by isoproterenol 10 nmol/L in a concen tration-dependent manner with the IC50 of 13.06 μmol/L. Similar to adenosine,AMP579 also showed an inhibitory effect on the ICa-L induced by isoproterenol.AMP579 and adenosine (both in 10 μmol/L) suppressed isoproterenol-induced ICa-L by 11.1% and 5.2%, respectively. In addition, AMP579 had a direct inhibitory effect on basal ICa-L in a concentration-dependent manner with IC50 (1.17 μmol/L).PD116948 (30 μmol/L), an adenosine A1 receptor blocker, showed no action on the inhibitory effect of AMP579 on basal ICa-L. However, GF109203X (0.4 μmol/L), a special protein kinase C (PKC) blocker, could abolish the inhibitory effect of AMP579 on basal ICa-L. So the inhibitory effect of AMP579 on basal ICa-L was induced through activating PKC, but not linked to adenosine A1 receptor. Conclusion:AMP579 shows a stronger inhibitory effect than adenosine on the ICa-L induced by isoproterenol. AMP579 also has a strong inhibitory effect on basal ICa-L in rat ventricular myocytes. Activation of PKC is involved in the inhibitory effect of AMP579 on basal ICa-L at downstream-mechanism.

  14. Down-regulation of the A3 adenosine receptor in human mast cells upregulates mediators of angiogenesis and remodeling.

    Rudich, Noam; Dekel, Ornit; Sagi-Eisenberg, Ronit


    Adenosine activated mast cells have been long implicated in allergic asthma and studies in rodent mast cells have assigned the A3 adenosine receptor (A3R) a primary role in mediating adenosine responses. Here we analyzed the functional impact of A3R activation on genes that are implicated in tissue remodeling in severe asthma in the human mast cell line HMC-1 that shares similarities with lung derived human mast cells. Quantitative real time PCR demonstrated upregulation of IL6, IL8, VEGF, amphiregulin and osteopontin. Moreover, further upregulation of these genes was noted upon the addition of dexamethasone. Unexpectedly, activated A3R down regulated its own expression and knockdown of the receptor replicated the pattern of agonist induced gene upregulation. This study therefore identifies the human mast cell A3R as regulator of tissue remodeling gene expression in human mast cells and demonstrates a heretofore-unrecognized mode of feedback regulation that is exerted by this receptor.

  15. Detection of ouabain-insensitive H(+)-transporting, K(+)-stimulated p-nitrophenylphosphatase activity in rat gastric glands by cerium-based cytochemistry.

    Kobayashi, T; Seguchi, H


    We employed a modification of our previously reported cerium-based cytochemical method for ouabain-sensitive, K-dependent p-nitrophenylphosphatase (Na-K ATPase) activity to detect ouabain-insensitive, K-stimulated p-nitrophenylphosphatase (K-pNPPase) activity in rat gastric glands. Biochemically, the enzyme activity of gastric glands incubated in a medium containing 50 mM Tricine buffer (pH 7.5), 50 mM KCl, 10 mM MgCl2, 2 mM CeCl3, 2 mM p-nitrophenylphosphate (pNPP), 2.5 mM levamisole, 10 mM ouabain, and 0.00015% Triton X-100, was optimal at pH 7.5-8.0 and decreased above pH 8.5. The amount of p-nitrophenol after incubation increased linearly in proportion to the amount of tissue in the medium. The enzyme activity was inhibited by omeprazole, sodium flouride (NaF), N-ethylmaleimide (NEM), and dicyclohexylcarbodiimide (DCCD). Heat-treated specimens had no enzyme activity. The enzyme activity increased with addition of K ions up to the concentration of 50 mM, and became constant above 50 mM. Cytochemically, the parietal cells of the gastric glands reacted positively for ouabain-insensitive K-pNPPase activity. Intense reaction was observed at the microvilli of the luminal surface and the intracellular canaliculi. The tubulovesicular system showed weak enzyme activity. The reaction products were found as fine, granular, electron-dense deposits in the cytoplasm just beneath the plasma membrane. The ouabain-insensitive K-pNPPase activity detected in this study appears, therefore, to be associated with that of H-transporting, K-stimulated adenosine triphosphatase (H-K ATPase).

  16. Adenosine-5'-phosphosulfate kinase is essential for Arabidopsis viability.

    Mugford, Sarah G; Matthewman, Colette A; Hill, Lionel; Kopriva, Stanislav


    In Arabidopsis thaliana, adenosine-5'-phosphosulfate kinase (APK) provides activated sulfate for sulfation of secondary metabolites, including the glucosinolates. We have successfully isolated three of the four possible triple homozygous mutant combinations of this family. The APK1 isoform alone was sufficient to maintain WT levels of growth and development. Analysis of apk1 apk2 apk3 and apk1 apk3 apk4 mutants suggests that APK3 and APK4 are functionally redundant, despite being located in cytosol and plastids, respectively. We were, however, unable to isolate apk1 apk3 apk4 mutants, most probably because the apk1 apk3 apk4 triple mutant combination is pollen lethal. Therefore, we conclude that APS kinase is essential for plant reproduction and viability.

  17. The adenosine A2B receptor is involved in anion secretion in human pancreatic duct Capan-1 epithelial cells

    Hayashi, M.; Inagaki, A.; Novak, Ivana


    by CFTRinh-172, a cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel inhibitor. The adenosine A2B receptor agonist, BAY 60-6583, increased Isc and whole-cell Cl− currents through CFTR Cl− channels, whereas the A2A receptor agonist, CGS 21680, had negligible effects. The A2B receptor....... These results demonstrate that luminal adenosine regulates anion secretion by activating CFTR Cl− channels via adenosine A2B receptors on the luminal membranes of Capan-1 cells. The present study endorses that purinergic signaling is important in the regulation of pancreatic secretion....

  18. [Concentration of prostaglandins and cyclic adenosine-3',5'-monophosphate in the tissues of rats].

    Komissarenko, V P; Slavnov, V N; Epsheĭn, E V; Malinkovich, V D


    The content of prostaglandines (PG) and cyclic 3',5'-adenosine monphosphate (cAMP) was investigated in rat tissues by the radioisotopic method of competitive binding. Maximum quantities of both PG and cAMP were revealed in the same most actively functioning organs: the brain, incretory glands, small intestine. Fatty tissue showed minimum quantities of these substances. Results indicate a close functional relationship between the PG synthesis and adenylatecyclase activity in the body tissues.

  19. Endogenous adenosine curtails lipopolysaccharide-stimulated tumour necrosis factor synthesis

    Eigler, A; Greten, T F; Sinha, B; Haslberger, C; Sullivan, G W; Endres, S


    Recent studies have demonstrated the inhibitory effect of exogenous adenosine on TNF production. During inflammation endogenous adenosine levels are elevated and may be one of several anti-inflammatory mediators that reduce TNF synthesis. In the present study the authors investigated this role of ad

  20. Synthesis and anti-renal fibrosis activity of conformationally locked truncated 2-hexynyl-N(6)-substituted-(N)-methanocarba-nucleosides as A3 adenosine receptor antagonists and partial agonists.

    Nayak, Akshata; Chandra, Girish; Hwang, Inah; Kim, Kyunglim; Hou, Xiyan; Kim, Hea Ok; Sahu, Pramod K; Roy, Kuldeep K; Yoo, Jakyung; Lee, Yoonji; Cui, Minghua; Choi, Sun; Moss, Steven M; Phan, Khai; Gao, Zhan-Guo; Ha, Hunjoo; Jacobson, Kenneth A; Jeong, Lak Shin


    Truncated N(6)-substituted-(N)-methanocarba-adenosine derivatives with 2-hexynyl substitution were synthesized to examine parallels with corresponding 4'-thioadenosines. Hydrophobic N(6) and/or C2 substituents were tolerated in A3AR binding, but only an unsubstituted 6-amino group with a C2-hexynyl group promoted high hA2AAR affinity. A small hydrophobic alkyl (4b and 4c) or N(6)-cycloalkyl group (4d) showed excellent binding affinity at the hA3AR and was better than an unsubstituted free amino group (4a). A3AR affinities of 3-halobenzylamine derivatives 4f-4i did not differ significantly, with Ki values of 7.8-16.0 nM. N(6)-Methyl derivative 4b (Ki = 4.9 nM) was a highly selective, low efficacy partial A3AR agonist. All compounds were screened for renoprotective effects in human TGF-β1-stimulated mProx tubular cells, a kidney fibrosis model. Most compounds strongly inhibited TGF-β1-induced collagen I upregulation, and their A3AR binding affinities were proportional to antifibrotic effects; 4b was most potent (IC50 = 0.83 μM), indicating its potential as a good therapeutic candidate for treating renal fibrosis.

  1. Adenosine Receptors: Expression, Function and Regulation

    Sandeep Sheth


    Full Text Available Adenosine receptors (ARs comprise a group of G protein-coupled receptors (GPCR which mediate the physiological actions of adenosine. To date, four AR subtypes have been cloned and identified in different tissues. These receptors have distinct localization, signal transduction pathways and different means of regulation upon exposure to agonists. This review will describe the biochemical characteristics and signaling cascade associated with each receptor and provide insight into how these receptors are regulated in response to agonists. A key property of some of these receptors is their ability to serve as sensors of cellular oxidative stress, which is transmitted by transcription factors, such as nuclear factor (NF-κB, to regulate the expression of ARs. Recent observations of oligomerization of these receptors into homo- and heterodimers will be discussed. In addition, the importance of these receptors in the regulation of normal and pathological processes such as sleep, the development of cancers and in protection against hearing loss will be examined.

  2. Modulatory effect of adenosine receptors on the ascending and descending neural reflex responses of rat ileum

    Schusdziarra Volker


    Full Text Available Abstract Background Adenosine is known to act as a neuromodulator by suppressing synaptic transmission in the central and peripheral nervous system. Both the release of adenosine within the small intestine and the presence of adenosine receptors on enteric neurons have been demonstrated. The aim of the present study was to characterize a possible involvement of adenosine receptors in the modulation of the myenteric reflex. The experiments were carried out on ileum segments 10 cm in length incubated in an single chambered organ bath, and the reflex response was initiated by electrical stimulation (ES. Results ES caused an ascending contraction and a descending relaxation followed by a contraction. All motility responses to ES were completely blocked by tetrodotoxin, indicating that they are mediated by neural mechanisms. Atropine blocked the contractile effects, whereas the descending relaxation was significantly increased. The A1 receptor agonist N6-cyclopentyladenosine increased the ascending contraction, whereas the ascending contraction was reduced by the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. Activation of the A1 receptor further reduced the descending relaxation and the latency of the peristaltic reflex. The A2B receptor antagonist alloxazine increased ascending contraction, whereas descending relaxation remained unchanged. For A2A and A3 receptors, we found contradictory effects of the agonists and antagonists, thus there is no clear physiological role for these receptors at this time. Conclusions This study suggests that the myenteric ascending and descending reflex response of the rat small intestine is modulated by release of endogenous adenosine via A1 receptors.

  3. Cloning of two adenosine receptor subtypes from mouse bone marrow-derived mast cells.

    Marquardt, D L; Walker, L L; Heinemann, S


    Adenosine potentiates the stimulated release of mast cell mediators. Pharmacologic studies suggest the presence of two adenosine receptors, one positively coupled to adenylate cyclase and the other coupled to phospholipase C activation. To identify mast cell adenosine receptor subtypes, cDNAs for the A1 and A2a adenosine receptors were obtained by screening a mouse brain cDNA library with the use of PCR-derived probes. Mouse bone marrow-derived mast cell cDNA libraries were constructed and screened with the use of A1 and A2a cDNA probes, which revealed the presence of A2a, but not A1, receptor clones. A putative A2b receptor was identified by using low stringency mast cell library screening. Northern blotting of mast cell poly(A)+ RNA with the use of receptor subtype probes labeled single mRNA bands of 2.4 kb and 1.8 kb for the A2a and A2b receptors, respectively. In situ cells. An A2a receptor-specific agonist failed to enhance mast cell mediator release, which suggests that the secretory process is modulated through the A2b and/or another receptor subtype. By using RNase protection assays, we found that mast cells that had been cultured in the presence of N-ethylcarboxamidoadenosine for 24 h exhibited a decrease in both A2a and A2b receptor RNA levels. Cells that had been cultured for 1 to 2 days in the presence of dexamethasone demonstrated increased amounts of A2a receptor mRNA, but no identifiable change in A2b receptor mRNA. Mast cells possess at least two adenosine receptor subtypes that may be differentially regulated.

  4. A High-Affinity Adenosine Kinase from Anopheles Gambiae

    M Cassera; M Ho; E Merino; E Burgos; A Rinaldo-Matthis; S Almo; V Schramm


    Genome analysis revealed a mosquito orthologue of adenosine kinase in Anopheles gambiae (AgAK; the most important vector for the transmission of Plasmodium falciparum in Africa). P. falciparum are purine auxotrophs and do not express an adenosine kinase but rely on their hosts for purines. AgAK was kinetically characterized and found to have the highest affinity for adenosine (K{sub m} = 8.1 nM) of any known adenosine kinase. AgAK is specific for adenosine at the nucleoside site, but several nucleotide triphosphate phosphoryl donors are tolerated. The AgAK crystal structure with a bound bisubstrate analogue Ap{sub 4}A (2.0 {angstrom} resolution) reveals interactions for adenosine and ATP and the geometry for phosphoryl transfer. The polyphosphate charge is partly neutralized by a bound Mg{sup 2+} ion and an ion pair to a catalytic site Arg. The AgAK structure consists of a large catalytic core in a three-layer {alpha}/{beta}/{alpha} sandwich, and a small cap domain in contact with adenosine. The specificity and tight binding for adenosine arise from hydrogen bond interactions of Asn14, Leu16, Leu40, Leu133, Leu168, Phe168, and Thr171 and the backbone of Ile39 and Phe168 with the adenine ring as well as through hydrogen bond interactions between Asp18, Gly64, and Asn68 and the ribosyl 2'- and 3'-hydroxyl groups. The structure is more similar to that of human adenosine kinase (48% identical) than to that of AK from Toxoplasma gondii (31% identical). With this extraordinary affinity for AgAK, adenosine is efficiently captured and converted to AMP at near the diffusion limit, suggesting an important role for this enzyme in the maintenance of the adenine nucleotide pool. mRNA analysis verifies that AgAK transcripts are produced in the adult insects.

  5. Common channels for water and protons at apical and basolateral cell membranes of frog skin and urinary bladder epithelia. Effects of oxytocin, heavy metals, and inhibitors of H(+)-adenosine triphosphatase

    Harvey, B.; Lacoste, I.; Ehrenfeld, J. (Commissariat a l' Energie Atomique, Villefranche-sur-mer (France))


    We have compared the response of proton and water transport to oxytocin treatment in isolated frog skin and urinary bladder epithelia to provide further insights into the nature of water flow and H+ flux across individual apical and basolateral cell membranes. In isolated spontaneous sodium-transporting frog skin epithelia, lowering the pH of the apical solution from 7.4 to 6.4, 5.5, or 4.5 produced a fall in pHi in principal cells which was completely blocked by amiloride, indicating that apical Na+ channels are permeable to protons. When sodium transport was blocked by amiloride, the H+ permeability of the apical membranes of principal cells was negligible but increased dramatically after treatment with antidiuretic hormone (ADH). In the latter condition, lowering the pH of the apical solution caused a voltage-dependent intracellular acidification, accompanied by membrane depolarization, and an increase in membrane conductance and transepithelial current. These effects were inhibited by adding Hg2+ (100 microM) or dicyclohexylcarbodiimide (DCCD, 10(-5) M) to the apical bath. Net titratable H+ flux across frog skin was increased from 30 +/- 8 to 115 +/- 18 (n = 8) after oxytocin treatment (at apical pH 5.5 and serosal pH 7.4) and was completely inhibited by DCCD (10(-5) M). The basolateral membranes of the principal cells in frog skin epithelium were found to be spontaneously permeable to H+ and passive electrogenic H+ transport across this membrane was not affected by oxytocin. Lowering the pH of the basolateral bathing solution (pHb) produced an intracellular acidification and membrane depolarization (and an increase in conductance when the normal dominant K+ conductance of this membrane was abolished by Ba2+ 1 mM). These effects of low pHb were blocked by micromolar concentrations of heavy metals (Zn2+, Ni2+, Co2+, Cd2+, and Hg2+).

  6. Identification and function of adenosine A3 receptor in afferent arterioles.

    Lu, Yan; Zhang, Rui; Ge, Ying; Carlstrom, Mattias; Wang, Shaohui; Fu, Yiling; Cheng, Liang; Wei, Jin; Roman, Richard J; Wang, Lei; Gao, Xichun; Liu, Ruisheng


    Adenosine plays an important role in regulation of renal microcirculation. All receptors of adenosine, A1, A2A, A2B, and A3, have been found in the kidney. However, little is known about the location and function of the A3 receptor in the kidney. The present study determined the expression and role of A3 receptors in mediating the afferent arteriole (Af-Art) response and studied the interaction of A3 receptors with angiotensin II (ANG II), A1 and A2 receptors on the Af-Art. We found that the A3 receptor expressed in microdissected isolated Af-Art and the mRNA levels of A3 receptor were 59% of A1. In the isolated microperfused Af-Art, A3 receptor agonist IB-MECA did not have a constrictive effect. Activation of A3 receptor dilated the preconstricted Af-Art by norepinephrine and blunted the vasoconstrictive effect of both adenosine A1 receptor activation and ANG II on the Af-Art, respectively. Selective A2 receptor antagonist (both A2A and A2B) had no effect on A3 receptor agonist-induced vasodilation, indicating that the dilatory effect of A3 receptor activation is not mediated by activation of A2 receptor. We conclude that the A3 receptor is expressed in the Af-Art, and activation of the A3 receptor dilates the Af-Art.

  7. In Vitro Functional Study of Rice Adenosine 5'-Phosphosulfate Kinase

    WANG De-zhen; CHEN Guo-guo; LU Lu-jia; JIANG Zhao-jun; RAO Yu-chun; SUN Mei-hao


    Sulfate can be activated by ATP sulfurylase and adenosine 5'-phosphosulfate kinase (APSK)in vivo. Recent studies suggested that APSK inArabidopsis thaliana regulated the partition between APS reduction and phosphorylation and its activity can be modulated by cellular redox status. In order to study regulation of APSK in rice (OsAPSK),OsAPSK1 gene was cloned and its activity was analyzed. OsAPSK1 C36 and C69 were found to be the conserved counterparts of C86 and C119, which involved in disulfide formation in AtAPSK.C36A/C69A OsAPSK1 double mutation was made by site directed mutagenesis. OsAPSK1 and its mutant were prokaryotically over-expressed and purified, and then assayed for APS phosphorylation activity. OsAPSK1 activity was depressed by oxidized glutathione, while the activity of its mutantwas not. Further studies in the case that oxidative stress will fluctuatein vivo3'-phosphoadenosine-5'-phosphosulfate content, and all APSK isoenzymes have similar regulation patterns are necessary to be performed.

  8. Tissue kallikrein reverses insulin resistance and attenuates nephropathy in diabetic rats by activation of phosphatidylinositol 3-kinase/protein kinase B and adenosine 5'-monophosphate-activated protein kinase signaling pathways.

    Yuan, Gang; Deng, Juanjuan; Wang, Tao; Zhao, Chunxia; Xu, Xizheng; Wang, Peihua; Voltz, James W; Edin, Matthew L; Xiao, Xiao; Chao, Lee; Chao, Julie; Zhang, Xin A; Zeldin, Darryl C; Wang, Dao Wen


    We previously reported that iv delivery of the human tissue kallikrein (HK) gene reduced blood pressure and plasma insulin levels in fructose-induced hypertensive rats with insulin resistance. In the current study, we evaluated the potential of a recombinant adeno-associated viral vector expressing the HK cDNA (rAAV-HK) as a sole, long-term therapy to correct insulin resistance and prevent renal damage in streptozotocin-induced type-2 diabetic rats. Administration of streptozotocin in conjunction with a high-fat diet induced systemic hypertension, diabetes, and renal damage in rats. Delivery of rAAV-HK resulted in a long-term reduction in blood pressure, and fasting plasma insulin was significantly lower in the rAAV-HK group than in the control group. The expression of phosphatidylinositol 3-kinase p110 catalytic subunit and the levels of phosphorylation at residue Thr-308 of Akt, insulin receptor B, and AMP-activated protein kinases were significantly decreased in organs from diabetic animals. These changes were significantly attenuated after rAAV-mediated HK gene therapy. Moreover, rAAV-HK significantly decreased urinary microalbumin excretion, improved creatinine clearance, and increased urinary osmolarity. HK gene therapy also attenuated diabetic renal damage as assessed by histology. Together, these findings demonstrate that rAAV-HK delivery can efficiently attenuate hypertension, insulin resistance, and diabetic nephropathy in streptozotocin-induced diabetic rats.

  9. 腺苷受体激动剂降低低氧大鼠肺动脉高压及对诱导型一氧化氮合酶-一氧化氮和肾素-血管紧张素的影响%Adenosine receptors agonists mitigated PAH of rats induced by chronic hypoxia through reduction of renin activity/angiotensin Ⅱ levels and increase of inducible nitric oxide synthase-nitric oxide levels

    谭建新; 黄秀兰; 王波; 方兴; 黄迪南


    Objective Recent studies showed that adenosine played important roles in vasodilation.This study aimed to investigate the effects of adenosine,its A1 and A2b receptor agonists on pulmonary artery hypertension (PAH) induced by chronic hypoxia in rats by continuously subcutaneous administration with an osmotic pump for 14 days,and to see if rennin angiotensin system and inducible nitric oxygen synthase (iNOS)/nitric oxide (NO) mediate the effects.Method Fifty-six male SD rats were randomly assigned to seven groups.Each group included eight rats.They were normoxic group,hypoxic group,adenosine-treated group [adenosine was administered at a dose of 150 μg(kg · min) under the hypoxic condition],adenosine A1 receptor agonist CPA-treated group [CPA was administered at a dose of 20 μg/(kg · min)under the hypoxic condition],CPA plus selective adenosine A1 antagonist DPCPX-treated group [CPA and DPCPX were administered simultaneously under the hypoxic condition,the dose of CPA was the same as the above,and the dose of DPCPX was 25 μg/(kg · min)],adenosine A2b receptor agonist NECA-treated group [NECA was administered at a dose of 30 μg/(kg · min) under the hypoxic condition],NECA plus selective adenosine A2b receptor antagonist MRS-treated group [NECA and MRS1754 were administered simultaneously under the hypoxic condition,the dose of NECA was the same as the above,and the dose of MRS1754 was 50 μg/(kg · min)].Osmotic pumps containing adenosine or selective adenosine A1 receptor agonist (CPA),or nonselective but potent adenosine A2b receptor agonist (NECA) were placed subcutaneously 7 days after hypoxia and continuously administered the agents for 14 days.Mean pulmonary artery pressure (mPAP) was detected after administration of the agents.Then blood samples were taken from heart for measurement of renin activity,angiotensin Ⅱ (Ang Ⅱ) and endothelin-1 (ET-1) concentration by radioimmunoassay,NO by measuring nitrate.Small pulmonary arteries were prepared for

  10. Expression of Drosophila adenosine deaminase in immune cells during inflammatory response.

    Novakova, Milena; Dolezal, Tomas


    Extra-cellular adenosine is an important regulator of inflammatory responses. It is generated from released ATP by a cascade of ectoenzymes and degraded by adenosine deaminase (ADA). There are two types of enzymes with ADA activity: ADA1 and ADGF/ADA2. ADA2 activity originates from macrophages and dendritic cells and is associated with inflammatory responses in humans and rats. Drosophila possesses a family of six ADGF proteins with ADGF-A being the main regulator of extra-cellular adenosine during larval stages. Herein we present the generation of a GFP reporter for ADGF-A expression by a precise replacement of the ADGF-A coding sequence with GFP using homologous recombination. We show that the reporter is specifically expressed in aggregating hemocytes (Drosophila immune cells) forming melanotic capsules; a characteristic of inflammatory response. Our vital reporter thus confirms ADA expression in sites of inflammation in vivo and demonstrates that the requirement for ADA activity during inflammatory response is evolutionary conserved from insects to vertebrates. Our results also suggest that ADA activity is achieved specifically within sites of inflammation by an uncharacterized post-transcriptional regulation based mechanism. Utilizing various mutants that induce melanotic capsule formation and also a real immune challenge provided by parasitic wasps, we show that the acute expression of the ADGF-A protein is not driven by one specific signaling cascade but is rather associated with the behavior of immune cells during the general inflammatory response. Connecting the exclusive expression of ADGF-A within sites of inflammation, as presented here, with the release of energy stores when the ADGF-A activity is absent, suggests that extra-cellular adenosine may function as a signal for energy allocation during immune response and that ADGF-A/ADA2 expression in such sites of inflammation may regulate this role.

  11. Forced Treadmill Exercise Prevents Spatial Memory Deficits in Aged Rats Probably Through the Activation of Na(+), K(+)-ATPase in the Hippocampus.

    Vanzella, Cláudia; Sanches, Eduardo Farias; Odorcyk, Felipe Kawa; Nicola, Fabrício; Kolling, Janaína; Longoni, Aline; Dos Santos, Tiago Marcon; Wyse, Angela Terezinha de Souza; Netto, Carlos Alexandre


    Regular physical activity has shown to improve the quality of life and to prevent age-related memory deficits. Memory processing requires proper regulation of several enzymes such as sodium-potassium adenosine triphosphatase (Na(+), K(+)-ATPase) and acetylcholinesterase (AChE), which have a pivotal role in neuronal transmission. The present study investigated the effects of a treadmill running protocol in young (3 months), mature (6 months) and aged (22 months) Wistar rats, on: (a) cognitive function, as assessed in the Water maze spatial tasks; (b) Na(+), K(+)-ATPase and AChE activities in the hippocampus following cognitive training alone or treadmill running combined with cognitive training. Animals of all ages were assigned to naïve (with no behavioral or exercise training), sedentary (non-exercised, with cognitive training) and exercised (20 min of daily running sessions, 3 times per week for 4 weeks and with cognitive training) groups. Cognition was assessed by reference and working memory tasks run in the Morris Water maze; 24 h after last session of behavioral testing, hippocampi were collected for biochemical analysis. Results demonstrated that: (a) a moderate treadmill running exercise prevented spatial learning and memory deficits in aged rats; (b) training in the Water maze increased both Na(+), K(+)-ATPase and AChE activities in the hippocampus of mature and aged rats; (c) aged exercised rats displayed an even further increase of Na(+), K(+)-ATPase activity in the hippocampus, (d) enzyme activity correlated with memory performance in aged rats. It is suggested that exercise prevents spatial memory deficits in aged rats probably through the activation of Na(+), K(+)-ATPase in the hippocampus.

  12. Increased Cortical Extracellular Adenosine Correlates with Seizure Termination

    Van Gompel, Jamie J.; Bower, Mark R.; Worrell, Gregory A.; Stead, Matt; Chang, Su-Youne; Goerss, Stephan J.; Kim, Inyong; Bennet, Kevin E.; Meyer, Fredric B.; Marsh, W. Richard; Blaha, Charles D.; Lee, Kendall H.


    Objective Seizures are currently defined by their electrographic features. However, neuronal networks are intrinsically dependent upon neurotransmitters of which little is known regarding their peri-ictal dynamics. Evidence supports adenosine as having a prominent role in seizure termination, as its administration can terminate and reduce seizures in animal models. Further, microdialysis studies in humans suggest adenosine is elevated peri-ictally, but the relationship to the seizure is obscured by its temporal measurement limitations. Because electrochemical techniques can provide vastly superior temporal resolution, we test the hypothesis that extracellular adenosine concentrations rise during seizure termination in an animal model and humans using electrochemistry. Methods White farm swine (n=45) were used in an acute cortical model of epilepsy and 10 human epilepsy patients were studied during intraoperative electrocorticography (Ecog). Wireless Instantaneous Neurotransmitter Concentration Sensor (WINCS) based fast scan cyclic voltametry (FSCV) and fixed potential amperometry were obtained utilizing an adenosine specific triangular waveform or biosensors respectively. Results Simultaneous Ecog and electrochemistry demonstrated an average adenosine rise of 260% compared to baseline at 7.5 ± 16.9 seconds with amperometry (n=75 events) and 2.6 ± 11.2 seconds with FSCV (n=15 events) prior to electrographic seizure termination. In agreement with these animal data, adenosine elevation prior to seizure termination in a human patient utilizing FSCV was also seen. Significance Simultaneous Ecog and electrochemical recording supports the hypothesis that adenosine rises prior to seizure termination, suggesting that adenosine itself may be responsible for seizure termination. Future work using intraoperative WINCS based FSCV recording may help to elucidate the precise relationship between adenosine and seizure termination. PMID:24483230

  13. Adenosine and Preexcitation Variants: Reappraisal of Electrocardiographic Changes.

    Ali, Hussam; Lupo, Pierpaolo; Foresti, Sara; De Ambroggi, Guido; Epicoco, Gianluca; Fundaliotis, Angelica; Cappato, Riccardo


    Intravenous adenosine is a short-acting blocker of the atrioventricular node that has been used to unmask subtle or latent preexcitation, and also to enable catheter ablation in selected patients with absent or intermittent preexcitation. Depending on the accessory pathway characteristics, intravenous adenosine may produce specific electrocardiographic changes highly suggestive of the preexcitation variant. Herein, we view different ECG responses to this pharmacological test in various preexcitation patterns that were confirmed by electrophysiological studies. Careful analysis of electrocardiographic changes during adenosine test, with emphasis on P-delta interval, preexcitation degree, and atrioventricular block, can be helpful to diagnose the preexcitation variant/pattern.

  14. Non-additive modulation of synaptic transmission by serotonin, adenosine, and cholinergic modulators in the sensory thalamus

    Ya-Chin eYang


    Full Text Available The thalamus relays sensory information to the cortex. Oscillatory activities of the thalamocortical network are modulated by monoamines, acetylcholine, and adenosine, and could be the key features characteristic of different vigilance states. Although the thalamus is almost always subjective to the actions of more than just one neuromodulator, reports on the modulatory effect of coexisting neuromodulators on thalamic synaptic transmission are unexpectedly scarce. We found that either monoamine or adenosine decreases retinothalamic synaptic strength and short-term depression, whereas cholinergic modulators generally enhance postsynaptic response to presynaptic activity. However, combinations of different modulators tend to produce non-additive effect, not predictable based on the action of one single modulator. Acetylcholine, acting via nicotinic receptors, can interact with either serotonin or adenosine to abolish most short-term synaptic depression. Moreover, the coexistence of adenosine and monoamine, with or without acetylcholine, results in robustly decreased synaptic strength and transforms short-term synaptic depression to facilitation. These findings are consistent with a view that acetylcholine is essential for an enriched sensory flow through the thalamus, and the flow is trimmed down by concomitant monoamine or adenosine (presumably for the wakefulness and rapid-eye movement, or REM, sleep state, respectively. In contrast, concomitant adenosine and monoamine would lead to a markedly deprived (and high-pass filtered sensory flow, and thus the dramatic decrease of monoamine may constitute the essential demarcation between non-REM and REM sleep. The collective actions of different neuromodulators on thalamic synaptic transmission thus could be essential for the understanding of network responsiveness in different vigilance states.

  15. Synergistic myoprotection of L-arginine and adenosine in a canine model of global myocardial ischaemic reperfusion injury

    DU Lei; DIAN Ke; CHEN Hui-jiao; AN Qi; JIA Meng-xing; YANG Ping-liang; WANG Wei; DENG Shuo-zeng; LIU Jin


    Background Endogenous nitric oxide and adenosine increase simultaneously to keep the balance of energy demand and supply when the oxygen supply is insufficient, which suggests that nitric oxide and adenosine might exert a synergistic myoprotection during tissue hypoxia. In this study, we tested this hypothesis utilizing a canine model of prolonged global myocardial ischaemic reperfusion injury.Methods In this double blind, controlled study, the hearts of 24 anaesthetized mongrel dogs were arrested for 2 hours with aortic cross clamping and blood cardioplegia. The treatment groups were those supplemented with 2 mmol/L L-arginine (ARG), supplemented with 1 mmol/L adenosine (ADO), ARG + ADO supplemented with both, and no supplementation (control) (n=6 in each group). Haemodynamics, biochemical indices, adenosine triphosphate (ATP) content and myeloperoxidase activities of myocardium were determined to evaluate myocardial injury. Statistical comparison was performed by two way ANOVA.Results Although the requirements for inotropic supports were higher, the cardiac outputs were lower in control group than in ARG, ADO and the combination groups. Plasma cardiac troponin I levels were higher and the areas of hydropic changes were larger in control group than in ARG and ADO groups. Combination of arginine and adenosine provided further myoprotection with respect to better cardiac performance, lower release of cardiac troponin I, and smaller areas of hydropic changes compared with ARG and ADO groups. ATP content was higher, but myeloperoxidase activities of myocardium were significantly lower in the combination group than in control, ARG and ADO groups (P<0.05).Conclusions Combination of L-arginine and adenosine provides synergistic myoprotection in a canine model of global myocardial ischaemia. Thus, the combination is recommended when the heart is exposed to a prolonged ischaemia during cardiac surgery.

  16. Adenosine receptors located in the NTS contribute to renal sympathoinhibition during hypotensive phase of severe hemorrhage in anesthetized rats.

    Scislo, Tadeusz J; O'Leary, Donal S


    Stimulation of nucleus of the solitary tract (NTS) A(2a)-adenosine receptors elicits cardiovascular responses quite similar to those observed with rapid, severe hemorrhage, including bradycardia, hypotension, and inhibition of renal but activation of preganglionic adrenal sympathetic nerve activity (RSNA and pre-ASNA, respectively). Because adenosine levels in the central nervous system increase during severe hemorrhage, we investigated to what extent these responses to hemorrhage may be due to activation of NTS adenosine receptors. In urethane- and alpha-chloralose-anesthetized male Sprague-Dawley rats, rapid hemorrhage was performed before and after bilateral nonselective or selective blockade of NTS adenosine-receptor subtypes [A(1)- and A(2a)-adenosine-receptor antagonist 8-(p-sulfophenyl)theophylline (1 nmol/100 nl) and A(2a)-receptor antagonist ZM-241385 (40 pmol/100 nl)]. The nonselective blockade reversed the response in RSNA (-21.0 +/- 9.6 Delta% vs. +7.3 +/- 5.7 Delta%) (where Delta% is averaged percent change from baseline) and attenuated the average heart rate response (change of -14.8 +/- 4.8 vs. -4.4 +/- 3.4 beats/min). The selective blockade attenuated the RSNA response (-30.4 +/- 5.2 Delta% vs. -11.1 +/- 7.7 Delta%) and tended to attenuate heart rate response (change of -27.5 +/- 5.3 vs. -15.8 +/- 8.2 beats/min). Microinjection of vehicle (100 nl) had no significant effect on the responses. The hemorrhage-induced increases in pre-ASNA remained unchanged with either adenosine-receptor antagonist. We conclude that adenosine operating in the NTS via A(2a) and possibly A(1) receptors may contribute to posthemorrhagic sympathoinhibition of RSNA but not to the sympathoactivation of pre-ASNA. The differential effects of NTS adenosine receptors on RSNA vs. pre-ASNA responses to hemorrhage supports the hypothesis that these receptors are differentially located/expressed on NTS neurons/synaptic terminals controlling different sympathetic outputs.

  17. Sleep-Wake Regulation and Its Impact on Working Memory Performance: The Role of Adenosine

    Carolin Franziska Reichert


    Full Text Available The sleep-wake cycle is regulated by a fine-tuned interplay between sleep-homeostatic and circadian mechanisms. Compelling evidence suggests that adenosine plays an important role in mediating the increase of homeostatic sleep pressure during time spent awake and its decrease during sleep. Here, we summarize evidence that adenosinergic mechanisms regulate not only the dynamic of sleep pressure, but are also implicated in the interaction of homeostatic and circadian processes. We review how this interaction becomes evident at several levels, including electrophysiological data, neuroimaging studies and behavioral observations. Regarding complex human behavior, we particularly focus on sleep-wake regulatory influences on working memory performance and underlying brain activity, with a specific emphasis on the role of adenosine in this interplay. We conclude that a change in adenosinergic mechanisms, whether exogenous or endogenous, does not only impact on sleep-homeostatic processes, but also interferes with the circadian timing system.

  18. N6-烷基-2-烷氧基腺苷化合物的合成及抗血小板凝集活性%Synthesis of 2-Alkoxy-N6-alkyl Adenosine Compounds and Their Anti-platelet Aggregation Activity

    吴兆军; 李顺来; 丁忠仁; 杜洪光


    Guanosine (1) as the starting material was protected by acetic anhydride to get 2',3',5'-tri-O-acetyl-guanosine (2), then chlorinated with phosphorus oxychloride to obtain 2-amino-6-chloro-9-(2',3',5'-tri-O-acetyl-β-D-ribofuranosyl)purine (3). Compound 3 was diazotized and hydrolyzed, subsequently reacted with several alkyl halides respectively to afford 2-alkoxy-6-chloro-9-(2',3',5'-tri-O-acetyl-β-D-ribofuranosyl)purine (4a~4c). 2-Alkoxy-N6-alkyl adenosine compounds 5a~5c were acquired by aminolysis and deprotection reaction of compounds 4a~4c. All compounds 5a~5c have not been reported so far. The structures of compounds 5a~5c were identified by 1H NMR, 13C NMR, IR and HRMS techniques. What is more, the anti-platelet aggregation rates for the final compounds were measured. At a concentration of 100 μmol/L, the test results of the biological activity of anti-platelet aggregation showed that N6-(4-methylbenzyl)-2-benzyloxy adenosine (5c3) and N6-(2-phenethyl)-2-benzyloxy adenosine (5c4) have a relatively low aggregation rate and have a certain anti-platelet aggregation activity.%以鸟瞟呤核苷(1)为原料,经羟基保护得到2’,3’,5’-三-O-乙酰基鸟嘌呤核苷(2),2与三氯氧磷反应得到2-氨基-6-氯-9-(2’,3’,5’-三-O-乙酰基-β-D-呋喃核糖)嘌呤(3),3经重氮化、水解和O-烷基化得到2-烷氧基-6-氯-9-(2’,3’,5’-三-O-乙酰基-β-D-呋喃核糖)嘌呤(4a~4c),4a~4c经胺解和水解脱保护反应得到12个未见报道的N6-烷基-2-烷氧基腺苷化合物5a~5c.化合物的结构经1HNMR,13C NMR,IR和HRMS等得到表征,同时对合成的N6-烷基-2-烷氧基腺苷化合物进行了抗血小板凝集活性测试.结果表明,在测试浓度为100 μmol/L时,N6-(4-甲基苄基)-2-苄氧基腺苷(5c3)和N6-(2-苯乙基)-2-苄氧基腺苷(5c4)具有相对较低的聚集率,具有一定的抗血小板凝集活性.

  19. Inhibition of uptake of adenosine into human blood platelets

    Lips, J.P.M.; Sixma, J.J.; Trieschnigg, A.C.


    Adenosine transport into human blood platelets is mediated by two independent systems with different affinities. Both systems transport only purine nucleosides and no pyrimidine nucleosides. In experiments with differently substituted purine nucleosides, purines and analogues, differences in carrier

  20. Adenosine Deaminase Deficiency – More Than Just an Immunodeficiency

    Kathryn Victoria Whitmore; Hubert Bobby Gaspar


    Adenosine deaminase (ADA) deficiency is best known as a form of severe combined immunodeficiency (SCID) which results from mutations in the gene encoding adenosine deaminase. Affected patients present with clinical and immunological manifestations typical of a severe combined immunodeficiency. Therapies are currently available that can that target these immunological disturbances and treated patients show varying degrees of clinical improvement. However, there is now a growing body of evidenc...

  1. Low-dose adenosine stress echocardiography: Detection of myocardial viability

    Djordjevic-Dikic, Ana; Ostojic, Miodrag; Beleslin, Branko; Nedeljkovic, Ivana; Stepanovic, Jelena; Stojkovic, Sinisa; Petrasinovic, Zorica; Nedeljkovic, Milan; Saponjski, Jovica; Giga, Vojislav


    Objective The aim of this study was to evaluate the diagnostic potential of low-dose adenosine stress echocardiography in detection of myocardial viability. Background Vasodilation through low dose dipyridamole infusion may recruit contractile reserve by increasing coronary flow or by increasing levels of endogenous adenosine. Methods Forty-three patients with resting dyssynergy, due to previous myocardial infarction, underwent low-dose adenosine (80, 100, 110 mcg/kg/min in 3 minutes intervals) echocardiography test. Gold standard for myocardial viability was improvement in systolic thickening of dyssinergic segments of ≥ 1 grade at follow-up. Coronary angiography was done in 41 pts. Twenty-seven patients were revascularized and 16 were medically treated. Echocardiographic follow up data (12 ± 2 months) were available in 24 revascularized patients. Results Wall motion score index improved from rest 1.55 ± 0.30 to 1.33 ± 0.26 at low-dose adenosine (p < 0.001). Of the 257 segments with baseline dyssynergy, adenosine echocardiography identified 122 segments as positive for viability, and 135 as necrotic since no improvement of systolic thickening was observed. Follow-up wall motion score index was 1.31 ± 0.30 (p < 0.001 vs. rest). The sensitivity of adenosine echo test for identification of viable segments was 87%, while specificity was 95%, and diagnostic accuracy 90%. Positive and negative predictive values were 97% and 80%, respectively. Conclusion Low-dose adenosine stress echocardiography test has high diagnostic potential for detection of myocardial viability in the group of patients with left ventricle dysfunction due to previous myocardial infarction. Low dose adenosine stress echocardiography may be adequate alternative to low-dose dobutamine test for evaluation of myocardial viability. PMID:12812523

  2. Granulocyte colony-stimulating factor and drugs elevating extracellular adenosine synergize to enhance haematopoietic reconstitution in irradiated mice

    Pospisil, M.; Hofer, M.; Netikova, J.; Hola, J.; Vacek, A. [Academy of Sciences of the Czech Republic, Inst. of Biophysics, Brno (Czech Republic); Znojil, V.; Vacha, J. [Masaryk Univ., Medical Faculty, Brno (Czech Republic)


    The activation of adenosine receptors has recently been demonstrated to stimulate haematopoiesis. In the present study, we investigated the ability of drugs elevating extracellular adenosine to influence curative effects of granulocyte colony-stimulating factor (G-CSF) in mice exposed to a sublethal dose of 4 Gy of {sup 60}Co radiation. Elevation of extracellular adenosine in mice was induced by the combined administration of dipyridamole, a drug inhibiting the cellular uptake of adenosine, and adenosine monophosphate (AMP), an adenosine prodrug. The effects of dipyridamole plus AMP, and G-CSF, administered either alone or in combination, were evaluated. The drugs were injected to mice in a 4-d treatment regimen starting on d 3 after irradiation and the haematopoietic response was evaluated on d 7, 10, 14, 18 and 24 after irradiation. While the effects of G-CSF on the late maturation stages of blood cells, appearing shortly after the completion of the treatment, were not influenced by dipyridamole plus AMP, positive effects of the combination therapy occurred in the post-irradiation recovery phase which is dependent on the repopulation of haematopoietic stem cells. This was indicated by the significant elevation of counts of granulocyte-macrophage progenitor cells (GM-CFC) and granulocytic cells in the bone marrow (d 14), of GM-CFC (d 14), granulocytic and erythroid cells (d 14 and 18) in the spleen, and of neutrophils (d 18), monocytes (d 14 and 18) and platelets (d 18) in the peripheral blood. These effects suggest that the repopulation potential of the combination therapy lies in a common multi-lineage cell population. The results of this study implicate the promising possibility to enhance the curative effects of G-CSF under conditions of myelosuppressive state induced by radiation exposure. (au) 43 refs.

  3. Low-dose adenosine stress echocardiography: Detection of myocardial viability

    Nedeljkovic Milan


    Full Text Available Abstract Objective The aim of this study was to evaluate the diagnostic potential of low-dose adenosine stress echocardiography in detection of myocardial viability. Background Vasodilation through low dose dipyridamole infusion may recruit contractile reserve by increasing coronary flow or by increasing levels of endogenous adenosine. Methods Forty-three patients with resting dyssynergy, due to previous myocardial infarction, underwent low-dose adenosine (80, 100, 110 mcg/kg/min in 3 minutes intervals echocardiography test. Gold standard for myocardial viability was improvement in systolic thickening of dyssinergic segments of ≥ 1 grade at follow-up. Coronary angiography was done in 41 pts. Twenty-seven patients were revascularized and 16 were medically treated. Echocardiographic follow up data (12 ± 2 months were available in 24 revascularized patients. Results Wall motion score index improved from rest 1.55 ± 0.30 to 1.33 ± 0.26 at low-dose adenosine (p Conclusion Low-dose adenosine stress echocardiography test has high diagnostic potential for detection of myocardial viability in the group of patients with left ventricle dysfunction due to previous myocardial infarction. Low dose adenosine stress echocardiography may be adequate alternative to low-dose dobutamine test for evaluation of myocardial viability.

  4. Direct Growth Graphene on Cu Nanoparticles by Chemical Vapor Deposition as Surface-Enhanced Raman Scattering Substrate for Label-Free Detection of Adenosine

    Xu, Shicai; Jiang, Shouzhen; Wang, Jihua; Wei, Jie; Xu, Shida; Liu, Hanping


    We present a graphene/Cu nanoparticle hybrids (G/CuNPs) system as a surface-enhanced Raman scattering (SERS) substrate for adenosine detection. The Cu nanoparticles wrapped around a monolayer graphene shell were directly synthesized on flat quartz by chemical vapor deposition in a mixture of methane and hydrogen. The G/CuNPs showed an excellent SERS enhancement activity for adenosine. The minimum detected concentration of the adenosine in serum was demonstrated as low as 5 nM, and the calibration curve showed a good linear response from 5 to 500 nM. The capability of SERS detection of adenosine in real normal human urine samples based on G/CuNPs was also investigated and the characteristic peaks of adenosine were still recognizable. The reproducible and the ultrasensitive enhanced Raman signals could be due to the presence of an ultrathin graphene layer. The graphene shell was able to enrich and fix the adenosine molecules, which could also efficiently maintain chemical and optical stability of G/CuNPs. Based...

  5. 1-(beta-D-Erythrofuranosyl)adenosine.

    Kline, Paul C; Zhao, Hongqiu; Noll, Bruce C; Oliver, Allen G; Serianni, Anthony S


    The title compound, also known as beta-erythroadenosine, C(9)H(11)N(5)O(3), (I), a derivative of beta-adenosine, (II), that lacks the C5' exocyclic hydroxymethyl (-CH(2)OH) substituent, crystallizes from hot ethanol with two independent molecules having different conformations, denoted (IA) and (IB). In (IA), the furanose conformation is (O)T(1)-E(1) (C1'-exo, east), with pseudorotational parameters P and tau(m) of 114.4 and 42 degrees, respectively. In contrast, the P and tau(m) values are 170.1 and 46 degrees, respectively, in (IB), consistent with a (2)E-(2)T(3) (C2'-endo, south) conformation. The N-glycoside conformation is syn (+sc) in (IA) and anti (-ac) in (IB). The crystal structure, determined to a resolution of 2.0 A, of a cocrystal of (I) bound to the enzyme 5'-fluorodeoxyadenosine synthase from Streptomyces cattleya shows the furanose ring in a near-ideal (O)E (east) conformation (P = 90 degrees and tau(m) = 42 degrees) and the base in an anti (-ac) conformation.

  6. Effects of Inula britannica Extracts on Biological Activities against Tetranychus cinnabarinus and Several Enzyme Systems in T.cinnabarinus%欧亚旋覆花提取物对朱砂叶螨生物活性及其体内几种酶活性的影响

    段丹丹; 王有年; 成军; 招嘉宁; 师光禄


    Acaricidal activities of the extracts from Inula britannica against Tetranychus cinnabarinus and then effects on several enzymes in T. cinnabarinus were evaluated under laboratory conditions. It was found that crude extracts from /. britannica with petroleum ether exhibited high acaricidal activities against T. cinnabarinus , and the corrected mortality in 24 h after treatment was 92.05% at concentration of 2 mg· mL-1. After a liquid-liquid partition from petroleum ether crude extracts with methanol, the petroleum ether extracts were separated into 38 fractions by column chromatography, and further tests for their acaricidal activities were conducted. Fraction 33, the main component of the resulting extracts, was found to possess the strongest acaricidal activity against T. cinnabarinus in all 38 fractions, and its corrected mortality against T. cinnabarinus was 90. 12% in 24 h. Moreover, GC-MS analysis revealed that chemical composition of fraction 33 was ethyl palmitate. Laboratory bioassay indicated that the corrected mortality of ethyl palmitate against T. cinnabarinus after treatment for 24h was 90. 61 % , and the mean lethal concentration ( LC50) was (1. 255 ± 0. 167 ) mg · mL-1 . In order to investigate mechanism of the toxic effect of ethyl palmitate on T. cinnabarinus, several important enzymes including glutathione S-transferase (GST), acetylcholinesterase ( AChE ) and Na +-adenosine triphosphatase ( Na + -ATPase) as well as total protein content in T. cinnabarinus were tested by the colorimetric method. The experimental results showed that after treatment with ethyl palmitate, total protein content in T. cinnabarinus obviously increased, and the activities of GSTs and AchE in T. cinnabarinus were strongly induced, while the activity of Na + -ATPase in T. cinnabarinus was inhibited, which could block the transmit of nerve, and eventually result in the death of the mite. These results indicated that Inula britannica extracts possessed high acaricidal

  7. Activation of extracellular signal-regulated kinases, NF-kappa B, and cyclic adenosine 5'-monophosphate response element-binding protein in lung neutrophils occurs by differing mechanisms after hemorrhage or endotoxemia.

    Abraham, E; Arcaroli, J; Shenkar, R


    Acute lung injury is frequently associated with sepsis or blood loss and is characterized by a proinflammatory response and infiltration of activated neutrophils into the lungs. Hemorrhage or endotoxemia result in activation of cAMP response element-binding protein (CREB) and NF-kappa B in lung neutrophils as well as increased expression of proinflammatory cytokines, such as TNF-alpha and macrophage-inflammatory peptide-2, by these cells. Activation of the extracellular regulated kinase (ERK) pathway occurs in stress responses and is involved in CREB activation. In the present experiments, hemorrhage or endotoxemia produced increased activation of mitogen-activated protein kinase kinase (MEK)1/2 and ERK2 (p42), but not of ERK1 (p44), in lung neutrophils. ERK1, ERK2, and MEK1/2 were not activated in peripheral blood neutrophils after hemorrhage or endotoxemia. Inhibition of xanthine oxidase led to further increase in the activation of MEK1/2 and ERK2 in lung neutrophils after hemorrhage, but not after endotoxemia. Alpha-adrenergic blockade before hemorrhage resulted in increased activation in lung neutrophils of MEK1/2, ERK1, ERK2, and CREB, but decreased activation of NF-kappa B. In contrast, alpha-adrenergic blockade before endotoxemia was associated with decreased activation of MEK1/2, ERK2, and CREB, but increased activation of NF-kappa B. Beta-adrenergic blockade before hemorrhage did not alter MEK1/2 or ERK1 activation in lung neutrophils, but decreased activation of ERK2 and CREB, while increasing activation of NF-kappa B. Beta-adrenergic inhibition before endotoxemia did not affect activation of MEK1/2, ERK1, ERK2, CREB, or NF-kappa B. These data indicate that the pathways leading to lung neutrophil activation after hemorrhage are different from those induced by endotoxemia.

  8. N-ethyl-carboxamide adenosine inhibits perioral dyskinesias induced by sulpiride + SKF 38393 in rabbits.

    Caporali, M G; Scotti de Carolis, A; Popoli, P


    A pattern of perioral dyskinesia was induced in adult male rabbits by concomitant stimulation of dopamine D1 receptors (SKF 38393) and blockade of dopamine D2 receptors (sulpiride). Rabbits treated with sulpiride (6 and 12.5 mg/kg i.v.) then, 90 min thereafter, with SKF 38393 (0.1, 1 and 10 mg/kg i.v.) showed a pattern of perioral dyskinesia characterized by compulsive and repetitive sniffing, licking and vacuous chewing. These effects were completely prevented by the administration of N-ethylcarboxamide adenosine (NECA), an A2 > A1 adenosine receptor agonist. The present results confirm that perioral dyskinesia is dependent on the activation of dopamine D1 receptors. They also show that, in order to induce perioral dyskinesia in rabbits, a concomitant blockade of dopamine D2 receptors is required. Finally, the antagonistic effect of NECA on the appearance of perioral movements confirms that adenosine receptors play a key role in the control of dopamine-mediated effects.

  9. Adenosine kinase inhibition protects against cranial radiation-induced cognitive dysfunction

    Munjal M Acharya


    Full Text Available Clinical radiation therapy for the treatment of CNS cancers leads to unintended and debilitating impairments in cognition. Radiation-induced cognitive dysfunction is long lasting, however, the underlying molecular and cellular mechanisms are still not well established. Since ionizing radiation causes microglial and astroglial activation, we hypothesized that maladaptive changes in astrocyte function might be implicated in radiation-induced cognitive dysfunction. Among other gliotransmitters, astrocytes control the availability of adenosine, an endogenous neuroprotectant and modulator of cognition, via metabolic clearance through adenosine kinase (ADK. Adult rats exposed to cranial irradiation (10 Gy showed significant declines in performance of hippocampal-dependent cognitive function tasks (novel place recognition, novel object recognition, and contextual fear conditioning 1 month after exposure to ionizing radiation using a clinically relevant regimen. Irradiated rats spent less time exploring a novel place or object. Cranial irradiation also led to reduction in freezing behavior compared to controls in the fear conditioning task. Importantly, immunohistochemical analyses of irradiated brains showed significant elevation of ADK immunoreactivity in the hippocampus that was related to astrogliosis and increased expression of glial fibrillary acidic protein (GFAP. Conversely, rats treated with the ADK inhibitor 5-iodotubercidin (5-ITU, 3.1 mg/kg, i.p., for 6 days prior to cranial irradiation showed significantly improved behavioral performance in all cognitive tasks 1 month post exposure. Treatment with 5-ITU attenuated radiation-induced astrogliosis and elevated ADK immunoreactivity in the hippocampus. These results confirm an astrocyte-mediated mechanism where preservation of extracellular adenosine can exert neuroprotection also against radiation-induced pathology. These innovative findings link radiation-induced changes in cognition and CNS

  10. Hydroxycarbamide modulates components involved in the regulation of adenosine levels in blood cells from sickle-cell anemia patients.

    Silva-Pinto, Ana C; Dias-Carlos, Carolina; Saldanha-Araujo, Felipe; Ferreira, Flávia I S; Palma, Patrícia V B; Araujo, Amélia G; Queiroz, Regina H C; Elion, Jacques; Covas, Dimas T; Zago, Marco A; Panepucci, Rodrigo A


    Recent studies have demonstrated the role of adenosine (ADO) in sickle-cell anemia (SCA). ADO is produced by CD39 and CD73 and converted to inosine by adenosine deaminase (ADA). We evaluated the effects of hydroxycarbamide (HU) treatment on the modulation of adenosine levels in SCA patients. The expressions of CD39, CD73, and CD26 were evaluated by flow cytometry on blood cells in 15 HU-treated and 17 untreated patients and 10 healthy individuals. RNA was extracted from monocytes, and ADA gene expression was quantified by real-time PCR. ADA activity was also evaluated. We found that ADA transcripts were two times higher in monocytes of HU-treated patients, compared with untreated (P = 0.039). Monocytes of HU-treated patients expressed CD26, while monocytes of controls and untreated patients did not (P = 0.023). In treated patients, a lower percentage of T lymphocytes expressed CD39 compared with untreated (P = 0.003), and the percentage of T regulatory (Treg) cells was reduced in the treated group compared with untreated (P = 0.017) and controls (P = 0.0009). Besides, HU-treated patients displayed increased ADA activity, compared with untreated. Our results indicate a novel mechanism of action of HU mediated by the reduction of adenosine levels and its effects on pathophysiological processes in SCA.

  11. Determination of serum adenosine deaminase and xanthine oxidase levels in patients with crimean-congo hemorrhagic fever

    V. Kenan Celik


    Full Text Available OBJECTIVE: Crimean-Congo hemorrhagic fever is an acute viral hemorrhagic fever with a high mortality rate. Despite increasing knowledge about hemorrhagic fever viruses, little is known about the pathogenesis of Crimean-Congo hemorrhagic fever. In this study, we measured serum adenosine deaminase and xanthine oxidase levels in Crimean-Congo hemorrhagic fever patients. METHODS: Serum adenosine deaminase levels were measured with a sensitive colorimetric method described by Giusti and xanthine oxidase levels by the method of Worthington in 30 consecutive hospitalized patients (mean age 42.6 ± 21.0. Laboratory tests confirmed their diagnoses of Crimean-Congo hemorrhagic fever. Thirty-five subjects (mean age 42.9 ± 19.1 served as the control group. RESULTS: There was a significant difference in adenosine deaminase and xanthine oxidase levels between cases and controls (p0.05. CONCLUSION: Adenosine deaminase and xanthine oxidase levels were increased in patients with Crimean-Congo hemorrhagic fever. Elevated serum xanthine oxidase activity in patients with Crimean-Congo hemorrhagic fever may be associated with reactive oxygen species generated by the xanthine/xanthine oxidase system during inflammatory responses. In addition, elevated lipid peroxidation may contribute to cell damage and hemorrhage. The association of cell damage and hemorrhage with xanthine oxidase activity should be further investigated in large-scale studies.

  12. Phosphodiesterase 2 negatively regulates adenosine-induced transcription of the tyrosine hydroxylase gene in PC12 rat pheochromocytoma cells.

    Makuch, Edyta; Kuropatwa, Marianna; Kurowska, Ewa; Ciekot, Jaroslaw; Klopotowska, Dagmara; Matuszyk, Janusz


    Adenosine induces expression of the tyrosine hydroxylase (TH) gene in PC12 cells. However, it is suggested that atrial natriuretic peptide (ANP) inhibits expression of this gene. Using real-time PCR and luciferase reporter assays we found that ANP significantly decreases the adenosine-induced transcription of the TH gene. Results of measurements of cyclic nucleotide concentrations indicated that ANP-induced accumulation of cGMP inhibits the adenosine-induced increase in cAMP level. Using selective phosphodiesterase 2 (PDE2) inhibitors and a synthetic cGMP analog activating PDE2, we found that PDE2 is involved in coupling the ANP-triggered signal to the cAMP metabolism. We have established that ANP-induced elevated levels of cGMP as well as cGMP analog stimulate hydrolytic activity of PDE2, leading to inhibition of adenosine-induced transcription of the TH gene. We conclude that ANP mediates negative regulation of TH gene expression via stimulation of PDE2-dependent cAMP breakdown in PC12 cells.

  13. Adenosine A1 Receptor Suppresses Tonic GABAA Receptor Currents in Hippocampal Pyramidal Cells and in a Defined Subpopulation of Interneurons.

    Rombo, Diogo M; Dias, Raquel B; Duarte, Sofia T; Ribeiro, Joaquim A; Lamsa, Karri P; Sebastião, Ana M


    Adenosine is an endogenous neuromodulator that decreases excitability of hippocampal circuits activating membrane-bound metabotropic A1 receptor (A1R). The presynaptic inhibitory action of adenosine A1R in glutamatergic synapses is well documented, but its influence on inhibitory GABAergic transmission is poorly known. We report that GABAA receptor (GABAAR)-mediated tonic, but not phasic, transmission is suppressed by A1R in hippocampal neurons. Adenosine A1R activation strongly inhibits GABAAR agonist (muscimol)-evoked currents in Cornu Ammonis 1 (CA1) pyramidal neurons and in a specific subpopulation of interneurons expressing axonal cannabinoid receptor type 1. In addition, A1R suppresses tonic GABAAR currents measured in the presence of elevated ambient GABA as well as in naïve slices. The inhibition of GABAergic currents involves both protein kinase A (PKA) and protein kinase C (PKC) signaling pathways and decreases GABAAR δ-subunit expression. On the contrary, no A1R-mediated modulation was detected in phasic inhibitory postsynaptic currents evoked either by afferent electrical stimulation or by spontaneous quantal release. The results show that A1R modulates extrasynaptic rather than synaptic GABAAR-mediated signaling, and that this modulation selectively occurs in hippocampal pyramidal neurons and in a specific subpopulation of inhibitory interneurons. We conclude that modulation of tonic GABAAR signaling by adenosine A1R in specific neuron types may regulate neuronal gain and excitability in the hippocampus.

  14. Inhibitory effects of benzodiazepines on the adenosine A(2B) receptor mediated secretion of interleukin-8 in human mast cells.

    Hoffmann, Kristina; Xifró, Rosa Altarcheh; Hartweg, Julia Lisa; Spitzlei, Petra; Meis, Kirsten; Molderings, Gerhard J; von Kügelgen, Ivar


    The activation of adenosine A(2B) receptors in human mast cells causes pro-inflammatory responses such as the secretion of interleukin-8. There is evidence for an inhibitory effect of benzodiazepines on mast cell mediated symptoms in patients with systemic mast cell activation disease. Therefore, we investigated the effects of benzodiazepines on adenosine A(2B) receptor mediated interleukin-8 production in human mast cell leukaemia (HMC1) cells by an enzyme linked immunosorbent assay. The adenosine analogue N-ethylcarboxamidoadenosine (NECA, 0.3-3 μM) increased interleukin-8 production about 5-fold above baseline. This effect was attenuated by the adenosine A(2B) receptor antagonist MRS1754 (N-(4-cyanophenyl)-2-{4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy}-acetamide) 1 μM. In addition, diazepam, 4'-chlorodiazepam and flunitrazepam (1-30 μM) markedly reduced NECA-induced interleukin-8 production in that order of potency, whereas clonazepam showed only a modest inhibition. The inhibitory effect of diazepam was not altered by flumazenil 10 μM or PK11195 (1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide) 10 μM. Diazepam attenuated the NECA-induced expression of mRNA encoding for interleukin-8. Moreover, diazepam and flunitrazepam reduced the increasing effects of NECA on cAMP-response element- and nuclear factor of activated t-cells-driven luciferase reporter gene activities in HMC1 cells. Neither diazepam nor flunitrazepam affected NECA-induced increases in cellular cAMP levels in CHO Flp-In cells stably expressing recombinant human adenosine A(2B) receptors, excluding a direct action of benzodiazepines on human adenosine A(2B) receptors. In conclusion, this is the first study showing an inhibitory action of benzodiazepines on adenosine A(2B) receptor mediated interleukin-8 production in human mast (HMC1) cells. The rank order of potency indicates the involvement of an atypical benzodiazepine binding site.

  15. 辛伐他汀抑制肝星状细胞活化及其对腺苷酸活化蛋白激酶活性的影响%Simvastatin inhibits activation of hepatic stellate cells and promotes activation of adenosine monophosphate activated protein kinase

    曹伟; 闫蕾; 王玮; 赵彩彦


    the underlying molecular mechanism ofthe cholesterol-blocking drug,simvastatin,in treating nonalcoholic fatty liver fibrosis.Method A rat model of nonalcoholic fatty liver fibrosis was established by feeding Wistar rats a fat-rich diet.After treatment with simvastatin (4 mg/kg/day),liver histological specimens were stained with hematoxylin-eosin and Masson's trichrome for microscopic analysis.Expression of adenosine monophosphate-activated protein kinase-alpha (AMPKα) was evaluated by reverse transcription-polymerase chain reaction (RT-PCR; for mRNA) and Western blotting (protein).The levels of serum total cholesterol (TC),triglycerides (TG),alanine aminotransferase (ALT),aspartate aminotransferase (AST),and tumor necrosis factor-alpha (TNFa) were measured by standard biochemical assays.The human hepatic stellate cell line,LX-2 (quiescent or activated),was treated with transforming growth factor-beta l (TGF-β1) alone,simvastatin alone,or TGF-β1 +simvastatin.RT-PCR and Western blotting were used to determine changes in AMPKα mRNA and protein expression,respectively.Results In the rat model of nonalcoholic fatty liver fibrosis,the extent of pathological changes in hepatic tissues correlated with severity of disease progression.The levels of serum TC,TG,ALT,AST and TNFα were increased significantly in model rats (vs.healthy controls; all,P< 0.01).AMPKα mRNA expression and activity was significantly decreased in model rats (vs.healthy controls; P< 0.01 and P< 0.05,respectively).Simvastatin,treatment significantly improved all of these parameters in model rats (vs.untreated model rats; all,P< 0.05).In vitro simvastatin treatment of human HSCs significantly increased AMPKα activity (quiescent LX-2:0.93 -0.10 vs.0.72±0.09,activated LX-2:0.72±0.10 vs.0.54±0.10,q=7.00,6.00; all,P<0.01),decreased a-smooth muscle actin expression (mRNA:0.30±0.02 vs.0.36±0.02,protein:0.30±0.03 vs.0.38±0.02,q=11.245,11.216; all,P<0.01),and decreased collagen I expression

  16. An adenosine nucleoside analogue NITD008 inhibits EV71 proliferation.

    Shang, Luqing; Wang, Yaxin; Qing, Jie; Shu, Bo; Cao, Lin; Lou, Zhiyong; Gong, Peng; Sun, Yuna; Yin, Zheng


    Enterovirus 71 (EV71), one of the major causative agents of Hand-Foot-Mouth Disease (HFMD), causes severe pandemics and hundreds of deaths in the Asia-Pacific region annually and is an enormous public health threat. However, effective therapeutic antiviral drugs against EV71 are rare. Nucleoside analogues have been successfully used in the clinic for the treatment of various viral infections. We evaluated a total of 27 nucleoside analogues and discovered that an adenosine nucleoside analogue NITD008, which has been reported to be an antiviral reagent that specifically inhibits flaviviruses, effectively suppressed the propagation of different strains of EV71 in RD, 293T and Vero cells with a relatively high selectivity index. Triphosphorylated NITD008 (ppp-NITD008) functions as a chain terminator to directly inhibit the RNA-dependent RNA polymerase activity of EV71, and it does not affect the EV71 VPg uridylylation process. A significant synergistic anti-EV71 effect of NITD008 with rupintrivir (AG7088) (a protease inhibitor) was documented, supporting the potential combination therapy of NITD008 with other inhibitors for the treatment of EV71 infections.

  17. Intracellular Adenosine Triphosphate Deprivation through Lanthanide-Doped Nanoparticles.

    Tian, Jing; Zeng, Xiao; Xie, Xiaoji; Han, Sanyang; Liew, Oi-Wah; Chen, Yei-Tsung; Wang, Lianhui; Liu, Xiaogang


    Growing interest in lanthanide-doped nanoparticles for biological and medical uses has brought particular attention to their safety concerns. However, the intrinsic toxicity of this new class of optical nanomaterials in biological systems has not been fully evaluated. In this work, we systematically evaluate the long-term cytotoxicity of lanthanide-doped nanoparticles (NaGdF4 and NaYF4) to HeLa cells by monitoring cell viability (mitochondrial activity), adenosine triphosphate (ATP) level, and cell membrane integrity (lactate dehydrogenase release), respectively. Importantly, we find that ligand-free lanthanide-doped nanoparticles induce intracellular ATP deprivation of HeLa cells, resulting in a significant decrease in cell viability after exposure for 7 days. We attribute the particle-induced cell death to two distinct cell death pathways, autophagy and apoptosis, which are primarily mediated via the interaction between the nanoparticle and the phosphate group of cellular ATP. The understanding gained from the investigation of cytotoxicity associated with lanthanide-doped nanoparticles provides keen insights into the safe use of these nanoparticles in biological systems.

  18. Changes in the rate of formation and resistance to reabsorption of cerebrospinal fluid during deliberate hypotension induced with adenosine or hemorrhage.

    Shapira, Y; Artru, A A; Lam, A M


    Adenosine is recommended for induction of deliberate hypotension. Although its effects on brain vasculature and metabolism and intracranial pressure have been reported, its effects on cerebrospinal fluid dynamics have not. In this study the rate of cerebrospinal fluid formation (Vf), resistance to reabsorption of cerebrospinal fluid (Ra), and electroencephalogram (EEG) activity were determined in rabbits before and during decrease of cerebral perfusion pressure (CPP) with intravenous (iv) adenosine or hemorrhage. In the adenosine group (n = 6), Vf and Ra were determined at control CPP, at CPP of 50, 35, and 28 mmHg achieved with iv adenosine, and at CPP greater than 60 mmHg achieved with iv adenosine combined with iv phenylephrine. In the hemorrhage group (n = 6), Vf and Ra were determined at the first four experimental conditions only. Control values for Vf (9 +/- 3 and 9 +/- 4 microliter.min-1, mean +/- SD) and Ra (428 +/- 567 and 412 +/- 144 did not differ between groups. In the adenosine group, Vf did not change significantly when CPP was decreased. However, in the hemorrhage group, Vf decreased significantly at CPP of 50 and 35 mmHg and became unmeasurable at CPP of 28 mmHg. Ra did not change significantly in either group. An increase of low-frequency (0.5-3.0 Hz) EEG activity and/or decrease of higher-frequency (3.5-30 Hz) EEG activity occurred at CPP of 28 mmHg in the adenosine group and at CPP of 35 mmHg in the hemorrhage group.(ABSTRACT TRUNCATED AT 250 WORDS)

  19. Hepatitis C virus core protein induces energy metabolism disorders of hepatocytes by down-regulation of silent mating type information regulation 2 homolog-1 and adenosine monophosphate-acti vated protein kinase signaling pathway



    Objective To study the role of silent mating type information regulation2homotog-1(SIRT1)-adenosine monophosphate(AMP)-activated protein kinase(AMPK) signaling pathway in hepatitis C virus core protein(HCV-core)induced energy metabolism disorders

  20. Exposure to ethanol during neurodevelopment modifies crucial offspring rat brain enzyme activities in a region-specific manner.

    Stolakis, Vasileios; Liapi, Charis; Zarros, Apostolos; Kalopita, Konstantina; Memtsas, Vassilios; Botis, John; Tsagianni, Anastasia; Kimpizi, Despoina; Varatsos, Alexios; Tsakiris, Stylianos


    The experimental simulation of conditions falling within "the fetal alcohol spectrum disorder" (FASD) requires the maternal exposure to ethanol (EtOH) during crucial neurodevelopmental periods; EtOH has been linked to a number of neurotoxic effects on the fetus, which are dependent upon the extent and the magnitude of the maternal exposure to EtOH and for which very little is known with regard to the exact mechanism(s) involved. The current study has examined the effects of moderate maternal exposure to EtOH (10 % v/v in the drinking water) throughout gestation, or gestation and lactation, on crucial 21-day-old offspring Wistar rat brain parameters, such as the activities of acetylcholinesterase (AChE) and two adenosine triphosphatases (Na(+),K(+)-ATPase and Mg(2+)-ATPase), in major offspring CNS regions (frontal cortex, hippocampus, hypothalamus, cerebellum and pons). The implemented experimental setting has provided a comparative view of the neurotoxic effects of maternal exposure to EtOH between gestation alone and a wider exposure timeframe that better covers the human third trimester-matching CNS neurodevelopment period (gestation and lactation), and has revealed a CNS region-specific susceptibility of the examined crucial neurochemical parameters to the EtOH exposure schemes attempted. Amongst these parameters, of particular importance is the recorded extensive stimulation of Na(+),K(+)-ATPase in the frontal cortex of the EtOH-exposed offspring that seems to be a result of the deleterious effect of EtOH during gestation. Although this stimulation could be inversely related to the observed inhibition of AChE in the same CNS region, its dependency upon the EtOH-induced modulation of other systems of neurotransmission cannot be excluded and must be further clarified in future experimental attempts aiming to simulate and to shed more light on the milder forms of the FASD-related pathophysiology.

  1. Diagnostic Value of Adenosine Deaminase and Its Isoforms in Type II Diabetes Mellitus

    Bagher Larijani; Ramin Heshmat; Mina Ebrahimi-Rad; Shohreh Khatami; Shirin Valadbeigi; Reza Saghiri


    Background and Aims. In the present study, we have investigated the activity of adenosine deaminase (ADA) as a diagnostic marker in type 2 (or II) diabetes mellitus (T2DM). Design and Methods. The deaminase activity of ADA1 and ADA2 was determined in serum from 33 patients with type 2 (or II) diabetes mellitus and 35 healthy controls. We also determined the proportion of glycated hemoglobin (HbA1c). Results. Our results showed significant differences between total serum ADA (tADA) and ADA2 ac...

  2. Adenosine receptors in rat and human pancreatic ducts stimulate chloride transport

    Novak, Ivana; Hede, Susanne; Hansen, Mette


    these could be involved in secretory processes, which involve cystic fibrosis transmembrane regulator (CFTR) Cl(-) channels or Ca(2+)-activated Cl(-) channels and [Formula: see text] transporters. Reverse transcriptase polymerase chain reaction analysis on rat pancreatic ducts and human duct cell......, plasma membrane of many PANC-1 cells, but only a few CFPAC-1 cells. Taken together, our data indicate that A(2A) receptors open Cl(-) channels in pancreatic ducts cells with functional CFTR. We propose that adenosine can stimulate pancreatic secretion and, thereby, is an active player in the acini...

  3. Intracoronary adenosine improves myocardial perfusion in late reperfused myocardial infarction


    Background Myocardial perfusion associates with clinical syndromes and prognosis.Adenosine could improve myocardial perfusion of acute myocardial infarction within 6 hours,but few data are available on late perfusion of myocardial infarction (MI).This study aimed at quantitatively evaluating the value of intracoronary adenosine improving myocardial perfusion in late reperfused MI with myocardial contrast echocardiography(MCE).Methods Twenty-six patients with anterior wall infarcts were divided randomly into 2 groups:adenosine group(n=12) and normal saline group(n=14).Their history of myocardial infarction was about 3-12 weeks.Adenosine or normalsaline was given when the guiding wire crossed the lesion through percutaneous coronary intervention(PCI),then the balloon was dilated and stent(Cypher/Cypher select)was implanted at the lesion.Contrast pulse sequencing MCE with Sonovue contrast via the coronary route was done before PCI and 30 minutes after PCI.Video densitometry and contrast filled-blank area were calculated with the CUSQ off-line software.Heart function and cardiac events were followed up within 30 days.Results Perfusion in the segments of the criminal occlusive coronary artery in the adenosine group was better than that in the saline group(5.71±0.29 vs 4.95±1.22,P<0.05).Ischemic myocardial segment was deminished significantly afterPCI,but the meliorated area was bigger in the adenosine group than in the saline group((1.56±0.60)cm2 vs(1.02±0.56) cm2,P<0.05).The video densitometry in critical segments was also improved significantly in the adenosine group (5.53±0.36 vs 5.26±0.35,P<0.05).Left ventricular ejection fraction(LVEF)was improved in all patients after PCI,but EF was not significant between the two groups((67±6)% vs(62±7)%,P>0.05).There was no in-hospital or 30-day major adverse cardiac event(MACE)in the adenosine group but 3 MACE in the saline group in 30 days after PCI.Conclusions Adenosine could improve myocardial microvascular

  4. Functional expression of adenosine A2A and A3 receptors in the mouse dendritic cell line XS-106.

    Dickenson, John M; Reeder, Steve; Rees, Bob; Alexander, Steve; Kendall, Dave


    There is increasing evidence to suggest that adenosine receptors can modulate the function of cells involved in the immune system. For example, human dendritic cells derived from blood monocytes have recently been described to express functional adenosine A1, A2A and A3 receptors. Therefore, in the present study, we have investigated whether the recently established murine dendritic cell line XS-106 expresses functional adenosine receptors. The selective adenosine A3 receptor agonist 1-[2-chloro-6[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-beta-D-ribofuranuronamide (2-Cl-IB-MECA) inhibited forskolin-mediated [3H]cyclic AMP accumulation and stimulated concentration-dependent increases in p42/p44 mitogen-activated protein kinase (MAPK) phosphorylation. The selective adenosine A2A receptor agonist 4-[2-[[-6-amino-9-(N-ethyl-beta-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzene-propanoic acid (CGS 21680) stimulated a robust increase in [3H]cyclic AMP accumulation and p42/p44 MAPK phosphorylation. In contrast, the selective adenosine A1 receptor agonist CPA (N6-cyclopentyladenosine) did not inhibit forskolin-mediated [3H]cyclic AMP accumulation or stimulate increases in p42/p44 MAPK phosphorylation. These observations suggest that XS-106 cells express functional adenosine A2A and A3 receptors. The non-selective adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) inhibited lipopolysaccharide-induced tumour necrosis factor-alpha (TNF-alpha) release from XS-106 cells in a concentration-dependent fashion. Furthermore, treatment with Cl-IB-MECA (1 microM) or CGS 21680 (1 microM) alone produced a partial inhibition of lipopolysaccharide-induced TNF-alpha release (when compared to NECA), whereas a combination of both agonists resulted in the inhibition of TNF-alpha release comparable to that observed with NECA alone. Treatment of cells with the adenosine A2A receptor selective antagonists 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a

  5. Differential adenosine sensitivity of diaphragm and skeletal muscle arterioles.

    Aaker, Aaron; Laughlin, M H


    The hyperemic response in exercising skeletal muscle is dependent on muscle fiber-type composition and fiber recruitment patterns, but the vascular control mechanisms producing exercise hyperemia in skeletal muscle remain poorly understood. The purpose of this study was to test the hypothesis that arterioles from white, low-oxidative skeletal muscle are less responsive to adenosine-induced dilation than are arterioles from diaphragm (Dia) and red, high-oxidative skeletal muscle. Second-order arterioles (2As) were isolated from the white portion of gastrocnemius muscle (WG; low-oxidative, fast-twitch muscle tissue) and two types of high-oxidative skeletal muscle [Dia and red portion of gastrocnemius muscle (RG)] of rats. Results reveal that 2As from all three types of muscle dilated in response to the endothelium-dependent dilator acetylcholine (WG: 48 +/- 3%, Dia: 51 +/- 3%, RG: 74 +/- 3%). In contrast, adenosine dilated only 2As from WG (48 +/- 4%) and Dia (46 +/- 5%) but not those from RG (5 +/- 5%). Thus adenosine-induced dilator responses differed among 2As of these different types of muscle tissue. However, the results do not support our hypothesis because 2As from Dia and WG dilated in response to adenosine, whereas 2As from RG did not. We conclude that the adenosine responsiveness of 2As from rat skeletal muscle cannot be predicted only by the fiber-type composition or oxidative capacity of the skeletal muscle tissue wherein the arteriole lies.

  6. Caffeine-induced natriuresis and diuresis via blockade of hepatic adenosine-mediated sensory nerves and a hepatorenal reflex.

    Ming, Zhi; Lautt, W Wayne


    The hepatorenal reflex, activated by intrahepatic adenosine, is involved in the regulation of urine production in healthy rats and renal pathogenesis secondary to liver injury. Hepatic adenosine A1 receptors regulate the hepatorenal reflex. The aim of the present study was to evaluate whether caffeine mediates renal natriuresis and diuresis in healthy and diseased liver through this mechanism. Rats were anesthetized and instrumented to monitor systemic, hepatic, and renal circulation and urine production. Intrahepatic (intraportal but not intravenous) caffeine (5 mg·kg-1) increased urine flow (~82%) in healthy rats. This effect was abolished by liver denervation. Intraportal infusion of adenosine decreased urine production, and this response was abolished by intraportal but not intravenous caffeine. Liver injury was induced by intraperitoneal injection of thioacetamide (500 mg·kg-1), and functional assessment was performed 24 h later. Liver injury was associated with lower (~30%) glomerular filtration rate, lower (~18%) renal arterial blood flow, and lower urine production. Intraportal but not intravenous caffeine improved basal urine production and renal ability to increase urine production in response to saline overload. The liver-dependent diuretic effect of caffeine is consistent with the hypothesis for the adenosine-mediated mechanism of hepatorenal syndrome.

  7. Simultaneous determination of adenine,uridine and adenosine in cordyceps sinensis and its substitutes by LC/ESI-MS

    黄兰芳; 吴名剑; 孙贤军; 郭方遒; 梁逸曾; 李晓如


    A simple, sensitive and reproducible high performance liquid chromatography-mass spectrometry coupled with electrospray ionization method for simultaneous separation and determination of adenine, adenosine and uridine was developed. The analytical column is a 2.0 mm× 150 mm Shimadzu VP-ODS column and volume fraction of the mobile phase is 86.5 %water, 12.0%methanol and 1.5%formic acid. 2-chloroadenosine was used as internal standard. Selective ion monitoring mode and selective ion monitoring ions at ratio of mass to electric charge of 136 for adenine, 268 for adenosine and 267 for uridine were chosen for quantitative analysis of the three active components. The results show that the regression equations and linear range are Y=0. 062X+0. 005 and 2.0 - 140.0μg · mL 1for adenine, Y=0. 049X+0. 004 and 4. 0 - 115.0 μg · mL-1 for uridine, Y=0. 154X+0. 014 and 1.0 - 125.0 μg · mL 1 for adenosine. The limits of detection are 0.6 μg · mL 1 for adenine, 1.0μg · mL-1 for uri dine and 0.2 μg · mL 1 for adenosine.The recoveries of the three constituents are from 96.6% to 103.2%.

  8. The effects of the adenosine A3 receptor agonist IB-MECA on sodium taurocholate-induced experimental acute pancreatitis.

    Prozorow-Krol, Beata; Korolczuk, Agnieszka; Czechowska, Grazyna; Slomka, Maria; Madro, Agnieszka; Celinski, Krzysztof


    The role of adenosine A3 receptors and their distribution in the gastrointestinal tract have been widely investigated. Most of the reports discuss their role in intestinal inflammations. However, the role of adenosine A3 receptor agonist in pancreatitis has not been well established. The aim of this study is [corrected] to evaluate the effects of the adenosine A3 receptor agonist on the course of sodium taurocholate-induced experimental acute pancreatitis (EAP). The experiments were performed on 80 male Wistar rats, 58 of which survived, subdivided into 3 groups: C--control rats, I--EAP group, and II--EAP group treated with the adenosine A3 receptor agonist IB-MECA (1-deoxy-1-6[[(3-iodophenyl) methyl]amino]-9H-purin-9-yl)-N-methyl-B-D-ribofuronamide at a dose of 0.75 mg/kg b.w. i.p. at 48, 24, 12 and 1 h before and 1 h after the injection of 5% sodium taurocholate solution into the biliary-pancreatic duct. Serum for α-amylase and lipase determinations and tissue samples for morphological examinations were collected at 2, 6, and 24 h of the experiment. In the IB-MECA group, α-amylase activity was decreased with statistically high significance compared to group I. The activity of lipase was not significantly different among the experimental groups but higher than in the control group. The administration of IB-MECA attenuated the histological parameters of inflammation as compared to untreated animals. The use of A3 receptor agonist IB-MECA attenuates EAP. Our findings suggest that stimulation of adenosine A3 receptors plays a positive role in the sodium taurocholate-induced EAP in rats.

  9. Caffeine acts via A1 adenosine receptors to disrupt embryonic cardiac function.

    Daniela L Buscariollo

    Full Text Available BACKGROUND: Evidence suggests that adenosine acts via cardiac A1 adenosine receptors (A1ARs to protect embryos against hypoxia. During embryogenesis, A1ARs are the dominant regulator of heart rate, and A1AR activation reduces heart rate. Adenosine action is inhibited by caffeine, which is widely consumed during pregnancy. In this study, we tested the hypothesis that caffeine influences developing embryos by altering cardiac function. METHODOLOGY/PRINCIPAL FINDINGS: Effects of caffeine and adenosine receptor-selective antagonists on heart rate were studied in vitro using whole murine embryos at E9.5 and isolated hearts at E12.5. Embryos were examined in room air (21% O(2 or hypoxic (2% O(2 conditions. Hypoxia decreased heart rates of E9.5 embryos by 15.8% and in E12.5 isolated hearts by 27.1%. In room air, caffeine (200 µM had no effect on E9.5 heart rates; however, caffeine increased heart rates at E12.5 by 37.7%. Caffeine abolished hypoxia-mediated bradycardia at E9.5 and blunted hypoxia-mediated bradycardia at E12.5. Real-time PCR analysis of RNA from isolated E9.5 and E12.5 hearts showed that A1AR and A2aAR genes were expressed at both ages. Treatment with adenosine receptor-selective antagonists revealed that SCH-58261 (A2aAR-specific antagonist had no affects on heart function, whereas DPCPX (A1AR-specific antagonist had effects similar to caffeine treatment at E9.5 and E12.5. At E12.5, embryonic hearts lacking A1AR expression (A1AR-/- had elevated heart rates compared to A1AR+/- littermates, A1AR-/- heart rates failed to decrease to levels comparable to those of controls. Caffeine did not significantly affect heart rates of A1AR-/- embryos. CONCLUSIONS/SIGNIFICANCE: These data show that caffeine alters embryonic cardiac function and disrupts the normal cardiac response to hypoxia through blockade of A1AR action. Our results raise concern for caffeine exposure during embryogenesis, particularly in pregnancies with increased risk of

  10. Effect of diazinon in male rats. Histopathological and biochemical studies.

    Dikshith, T S; Behari, J R; Datta, K K; Mathur, A K


    Mild structural and functional changes were observed in liver and testes of rats after a single intraperitoneal administration of diazinon (21.6 mg/kg). Kidney, however, showed no pathological lesion. Attempts are made to correlate the pathological changes in these organs with the activity of succinic dehydrogenase, adenosine triphosphatase and alkaline phosphatase.

  11. Adenosine triphosphate inhibits melatonin synthesis in the rat pineal gland.

    Souza-Teodoro, Luis Henrique; Dargenio-Garcia, Letícia; Petrilli-Lapa, Camila Lopes; Souza, Ewerton da Silva; Fernandes, Pedro A C M; Markus, Regina P; Ferreira, Zulma S


    Adenosine triphosphate (ATP) is released onto the pinealocyte, along with noradrenaline, from sympathetic neurons and triggers P2Y1 receptors that enhance β-adrenergic-induced N-acetylserotonin (NAS) synthesis. Nevertheless, the biotransformation of NAS into melatonin, which occurs due to the subsequent methylation by acetylserotonin O-methyltransferase (ASMT; EC, has not yet been evaluated in the presence of purinergic stimulation. We therefore evaluated the effects of purinergic signaling on melatonin synthesis induced by β-adrenergic stimulation. ATP increased NAS levels, but, surprisingly, inhibited melatonin synthesis in an inverse, concentration-dependent manner. Our results demonstrate that enhanced NAS levels, which depend on phospholipase C (PLC) activity (but not the induction of gene transcription), are a post-translational effect. By contrast, melatonin reduction is related to an ASMT inhibition of expression at both the gene transcription and protein levels. These results were independent of nuclear factor-kappa B (NF-kB) translocation. Neither the P2Y1 receptor activation nor the PLC-mediated pathway was involved in the decrease in melatonin, indicating that ATP regulates pineal metabolism through different mechanisms. Taken together, our data demonstrate that purinergic signaling differentially modulates NAS and melatonin synthesis and point to a regulatory role for ATP as a cotransmitter in the control of ASMT, the rate-limiting enzyme in melatonin synthesis. The endogenous production of melatonin regulates defense responses; therefore, understanding the mechanisms involving ASMT regulation might provide novel insights into the development and progression of neurological disorders since melatonin presents anti-inflammatory, neuroprotective, and neurogenic effects.

  12. Tween 20-stabilized gold nanoparticles combined with adenosine triphosphate-BODIPY conjugates for the fluorescence detection of adenosine with more than 1000-fold selectivity

    Hung, Szu-Ying; Shih, Ya-Chen [Department of Chemistry, National Sun Yat-sen University, Taiwan (China); Tseng, Wei-Lung, E-mail: [Department of Chemistry, National Sun Yat-sen University, Taiwan (China); School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Taiwan (China); Center for Nanoscience and Nanotechnology, National Sun Yat-sen University, Taiwan (China); Center for Stem Cell Research, Kaohsiung Medical University, Taiwan (China)


    Graphical abstract: A simple, enzyme-free, label-free, sensitive and selective system was developed for detecting adenosine based on the use of Tween 20-stabilized gold nanoparticles as an efficient quencher for boron dipyrromethene-conjugated adenosine 5′-triphosphate and as a recognition element for adenosine. - Highlights: • The proposed method can detect adenosine with more than 1000-fold selectivity. • The analysis of adenosine is rapid (∼6 min) using the proposed method. • This method provided better sensitivity for adenosine as compared to aptamer-based sensors. • This method can be applied for the determination of adenosine in urine. - Abstract: This study describes the development of a simple, enzyme-free, label-free, sensitive, and selective system for detecting adenosine based on the use of Tween 20-stabilized gold nanoparticles (Tween 20-AuNPs) as an efficient fluorescence quencher for boron dipyrromethene-conjugated adenosine 5′-triphosphate (BODIPY-ATP) and as a recognition element for adenosine. BODIPY-ATP can interact with Tween 20-AuNPs through the coordination between the adenine group of BODIPY-ATP and Au atoms on the NP surface, thereby causing the fluorescence quenching of BODIPY-ATP through the nanometal surface energy transfer (NSET) effect. When adenosine attaches to the NP surface, the attached adenosine exhibits additional electrostatic attraction to BODIPY-ATP. As a result, the presence of adenosine enhances the efficiency of AuNPs in fluorescence quenching of BODIPY-ATP. The AuNP-induced fluorescence quenching of BODIPY-ATP progressively increased with an increase in the concentration of adenosine; the detection limit at a signal-to-noise ratio of 3 for adenosine was determined to be 60 nM. The selectivity of the proposed system was more than 1000-fold for adenosine over any adenosine analogs and other nucleotides. The proposed system combined with a phenylboronic acid-containing column was successfully applied to the

  13. Neuroprotective effects of adenosine isolated from Cordyceps cicadae against oxidative and ER stress damages induced by glutamate in PC12 cells.

    Olatunji, Opeyemi J; Feng, Yan; Olatunji, Oyenike O; Tang, Jian; Ouyang, Zhen; Su, Zhaoliang; Wang, Dujun; Yu, Xiaofeng


    Glutamate has been proven to induce oxidative stress through the formation of reactive oxygen species (ROS) and increased calcium overload which results in neuronal injury, development of neurodegenerative diseases and death. Adenosine is one of the bioactive nucleosides found in Cordyceps cicadae and it has displayed several pharmacological activities including neuroprotection. In this study, the protective effects of adenosine from C. cicadae against glutamate-induce oxidative stress in PC12 cells were evaluated. The exposure of PC12 cells to glutamate (5mM) induced the formation of ROS, increased Ca(2+) influx, endoplasmic reticulum (ER) stress and up regulated the expression of pro-apoptotic factor Bax. However, pretreatment with adenosine markedly increased cell viability, decreased the elevated levels of ROS and Ca(2+) induced by glutamate. Furthermore adenosine increased the activities of GSH-Px and SOD, as well as retained mitochondria membrane potential (MMP), increased Bcl-2/Bax ratio, and reduced the expression of ERK, p38, and JNK. Overall, our results suggest that adenosine may be a promising potential therapeutic agent for the prevention and treatment of neurodegenerative disorders.

  14. Maternal caffeine intake during gestation and lactation down-regulates adenosine A1 receptor in rat brain from mothers and neonates.

    Lorenzo, A M; León, D; Castillo, C A; Ruiz, M A; Albasanz, J L; Martín, M


    Even though caffeine can be excreted in breast milk, few studies have analyzed the effect of maternal caffeine consumption during lactation on neonatal brain. In the present work pregnant rats were treated daily with 1 g/L of caffeine in their drinking water during pregnancy and/or lactation and the effect on adenosine A(1) receptor in brains from both lactating mothers and 15 days-old neonates was assayed using radioligand binding and real time PCR assays. Mothers receiving caffeine during gestational period developed motor activation in gestational days 8-10 which was associated with a significant decrease of total adenosine A(1) receptor number (84%). A similar decrease was detected in mothers treated with caffeine during lactation (76%) and throughout gestation and lactation (73%); this was accompanied by a significant decrease in mRNA level coding adenosine A(1) receptor (28%). In male neonates, adenosine A(1) receptor was also decreased after chronic caffeine exposure during gestation (80%), lactation (76%) and gestation plus lactation (80%). In female neonates, adenosine A(1) receptor tended to decrease in response to caffeine exposure although no significant variations were found. No variation in the level of mRNA coding adenosine A(1) receptor was detected in neonates in any case. Concerning adenosine A(2A) receptor, radioligand binding assays revealed that this receptor remains unaltered in maternal and neonatal brain in response to caffeine exposure. However, caffeine consumption during gestation and lactation evoked a significant decrease in mRNA level coding A(2A) receptor (32%) in mothers' brain.

  15. Amplified fluorescence detection of adenosine via catalyzed hairpin assembly and host-guest interactions between β-cyclodextrin polymer and pyrene.

    Huang, Haihua; Yang, Xiaohai; Wang, Kemin; Wang, Qing; Guo, Qiuping; Huang, Jin; Liu, Jianbo; Song, Chunxia


    Nowadays, enzyme-free nucleic acid-based signal amplification strategies are frequently utilized in the design of biosensors due to their excellent sensitivity. Developing more extended analytical methods is fundamental for basic studies in the biological and biomedical fields. Herein, we introduce an enzyme-free amplified detection strategy for the small molecule adenosine. The approach is based on adenosine-aptamer binding triggered catalyzed hairpin assembly and host-guest interactions between β-cyclodextrin polymer (β-CDP) and pyrene. Two hairpin probes (probe H1 and probe H2) and an aptamer-trigger/inhibitor duplex probe were employed in the system and the pyrene-labeled probe H1 was chosen as the signal unit. In the absence of adenosine, the aptamer-trigger was inhibited by the inhibitor strand. The hairpin probes were in the closed hairpin formation without activation of the trigger strand. Pyrene labeled at the 5'-termini of the single-stranded stem of probe H1 could be easily trapped in the hydrophobic cavity of β-CDP because of weak steric hindrance, leading to a significant fluorescence enhancement. Once the hairpin assembly was catalyzed by the adenosine-aptamer binding event, a hybridized DNA duplex H1/H2 was created continuously. Pyrene labeled at the 5'-termini of the DNA duplex H1/H2 finds it difficult to enter the cavity of β-CDP due to steric hindrance, leading to a weaker fluorescence signal. Thus, the target could be detected by this simple mix-and-detect amplification method without a need for expensive and perishable protein enzymes. As low as 42 nM of adenosine was detected by this assay, which is comparable to that of some reported colorimetric methods. Meanwhile, the proposed method was further successfully applied to detect adenosine in human serum samples, showing great potential for adenosine detection from complex fluids.

  16. Identification of Electronic and Structural Descriptors of Adenosine Analogues Related to Inhibition of Leishmanial Glyceraldehyde-3-Phosphate Dehydrogenase

    Norka B. H. Lozano


    Full Text Available Quantitative structure–activity relationship (QSAR studies were performed in order to identify molecular features responsible for the antileishmanial activity of 61 adenosine analogues acting as inhibitors of the enzyme glyceraldehyde 3-phosphate dehydrogenase of Leishmania mexicana (LmGAPDH. Density functional theory (DFT was employed to calculate quantum-chemical descriptors, while several structural descriptors were generated with Dragon 5.4. Variable selection was undertaken with the ordered predictor selection (OPS algorithm, which provided a set with the most relevant descriptors to perform PLS, PCR and MLR regressions. Reliable and predictive models were obtained, as attested by their high correlation coefficients, as well as the agreement between predicted and experimental values for an external test set. Additional validation procedures were carried out, demonstrating that robust models were developed, providing helpful tools for the optimization of the antileishmanial activity of adenosine compounds.

  17. Brain Na+, K+-ATPase Activity In Aging and Disease

    de Lores Arnaiz, Georgina Rodríguez; Ordieres, María Graciela López


    Na+/K+ pump or sodium- and potassium-activated adenosine 5’-triphosphatase (Na+, K+-ATPase), its enzymatic version, is a crucial protein responsible for the electrochemical gradient across the cell membranes. It is an ion transporter, which in addition to exchange cations, is the ligand for cardenolides. This enzyme regulates the entry of K+ with the exit of Na+ from cells, being the responsible for Na+/K+ equilibrium maintenance through neuronal membranes. This transport system couples the hydrolysis of one molecule of ATP to exchange three sodium ions for two potassium ions, thus maintaining the normal gradient of these cations in animal cells. Oxidative metabolism is very active in brain, where large amounts of chemical energy as ATP molecules are consumed, mostly required for the maintenance of the ionic gradients that underlie resting and action potentials which are involved in nerve impulse propagation, neurotransmitter release and cation homeostasis. Protein phosphorylation is a key process in biological regulation. At nervous system level, protein phosphorylation is the major molecular mechanism through which the function of neural proteins is modulted in response to extracellular signals, including the response to neurotransmitter stimuli. It is the major mechanism of neural plasticity, including memory processing. The phosphorylation of Na+, K+-ATPase catalytic subunit inhibits enzyme activity whereas the inhibition of protein kinase C restores the enzyme activity. The dephosphorylation of neuronal Na+, K+-ATPase is mediated by calcineurin, a serine / threonine phosphatase. The latter enzyme is involved in a wide range of cellular responses to Ca2+ mobilizing signals, in the regulation of neuronal excitability by controlling the activity of ion channels, in the release of neurotransmitters and hormones, as well as in synaptic plasticity and gene transcription. In the present article evidence showing Na+, K+-ATPase involvement in signaling pathways

  18. Development of coronary vasospasm during adenosine-stress myocardial perfusion CT imaging

    Nam, Jeong Gu; Choi, Seong Hoon; Kang, Byeong Seong; Bang, Min Aeo; Kwon, Woon Jeong [Dept. of Radiology, Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan (Korea, Republic of)


    Adenosine is a short-acting coronary vasodilator, and it is widely used during pharmacological stress myocardial perfusion imaging. It has a well-established safety profile, and most of its side effects are known to be mild and transient. Until now, coronary vasospasm has been rarely reported as a side effect of adenosine during or after adenosine stress test. This study reports a case of coronary vasospasm which was documented on stress myocardial perfusion CT imaging during adenosine stress test.

  19. Differential effects of the adenosine A1 receptor agonist adenosine amine congener on renal, femoral and carotid vascular conductance in preterm fetal sheep.

    Booth, Lindsea C; Tummers, Leonie; Jensen, Ellen C; Barrett, Carolyn J; Malpas, Simon C; Gunn, Alistair J; Bennet, Laura


    1. Adenosine A(1) receptor activation is critical for endogenous neuroprotection from hypoxia-ischaemia, raising the possibility that treatment with A(1) receptor agonists may be an effective physiological protection strategy for vulnerable preterm infants. However, the A(1) receptor can mediate unwanted systemic effects, including vasoconstriction of the afferent glomerular arteriole. There is limited information on whether this occurs at doses that improve cerebral perfusion in the immature brain. 2. Therefore, in the present study, we examined whether infusion of the selective A(1) receptor agonist adenosine amine congener (ADAC) is associated with reduced renal perfusion in chronically instrumented preterm (0.7 gestation) fetal sheep. In the present study, ADAC was given in successive doses of 2.5, 5.0 and 15.0 microg, 45 min apart. 3. Treatment with ADAC was associated with a marked reduction in renal vascular conductance (and blood flow), whereas carotid conductance was increased and there was no significant effect on femoral conductance. In contrast with the stable effects of increasing ADAC dose on vascular conductance, there was a significant dose-related fall in fetal heart rate and blood pressure. 4. In conclusion, these short-term data support the concern that A(1) receptor agonist infusion can selectively impair renal perfusion, even at low doses.

  20. DMPD: Shaping of monocyte and macrophage function by adenosine receptors. [Dynamic Macrophage Pathway CSML Database

    Full Text Available 17056121 Shaping of monocyte and macrophage function by adenosine receptors. Hasko ...tml) (.csml) Show Shaping of monocyte and macrophage function by adenosine receptors. PubmedID 17056121 Titl...e Shaping of monocyte and macrophage function by adenosine receptors. Authors Has

  1. Adenosine A(3) receptor-induced CCL2 synthesis in cultured mouse astrocytes

    Wittendorp, MC; Boddeke, HWGM; Biber, K


    During neuropathological conditions, high concentrations of adenosine are released, stimulating adenosine receptors in neurons and glial cells. It has recently been shown that stimulation of adenosine receptors in glial cells induces the release of neuroprotective substances such as NGF, S-100beta,

  2. The role of glial adenosine receptors in neural resilience and the neurobiology of mood disorders

    Calker, D; Biber, K


    Adenosine receptors were classified into A(1)- and A(2)-receptors in the laboratory of Bernd Hamprecht more than 25 years ago. Adenosine receptors are instrumental to the neurotrophic effects of glia cells. Both microglia and astrocytes release after stimulation via adenosine receptors factors that

  3. Estimation of adenosine triphosphate utilization of rat mast cells during and after anaphylactic histamine secretion

    Johansen, Torben


    Determination of the cellular content of adenosine triphosphate (ATP) and the rate of ATP-synthesis were used to estimate the cellular utilization of ATP in relation to anaphylactic histamine secretion. There was an increased rate of oxidative ATP-synthesis and a decreased cellular ATP content...... during the time period of histamine secretion and immediately after its completion. During secretion the additional ATP-utilization above the basal level of ATP-synthesis was 0.51 pmol/10(3) cells. 2.5 min after cell activation, the rate of additional ATP-utilization was 0.30 pmol/10(3) cells...

  4. Equilibrium and kinetic selectivity profiling on the human adenosine receptors.

    Guo, Dong; Dijksteel, Gabrielle S; van Duijl, Tirsa; Heezen, Maxime; Heitman, Laura H; IJzerman, Adriaan P


    Classical evaluation of target selectivity is usually undertaken by measuring the binding affinity of lead compounds against a number of potential targets under equilibrium conditions, without considering the kinetics of the ligand-receptor interaction. In the present study we propose a combined strategy including both equilibrium- and kinetics-based selectivity profiling. The adenosine receptor (AR) was chosen as a prototypical drug target. Six in-house AR antagonists were evaluated in a radioligand displacement assay for their affinity and in a competition association assay for their binding kinetics on three AR subtypes. One of the compounds with a promising kinetic selectivity profile was also examined in a [(35)S]-GTPγS binding assay for functional activity. We found that XAC and LUF5964 were kinetically more selective for the A1R and A3R, respectively, although they are non-selective in terms of their affinity. In comparison, LUF5967 displayed a strong equilibrium-based selectivity for the A1R over the A2AR, yet its kinetic selectivity thereon was less pronounced. In a GTPγS assay, LUF5964 exhibited insurmountable antagonism on the A3R while having a surmountable effect on the A1R, consistent with its kinetic selectivity profile. This study provides evidence that equilibrium and kinetic selectivity profiling can both be important in the early phases of the drug discovery process. Our proposed combinational strategy could be considered for future medicinal chemistry efforts and aid the design and discovery of different or even better leads for clinical applications.

  5. Searching Inhibitors of Adenosine Kinase by Simulation Methods

    ZHU Rui-Xin; ZHANG Xing-Long; DONG Xi-Cheng; CHEN Min-Bo


    Searching new inhibitors of adenosine kinase (AK) is still drawing attention of experimental scientists. A better and solid model is here proposed by means of simulation methods from different ways, the direct analysis of receptor itself, the conventional 3D-QSAR methods and the integration of docking method and the conventional QSAR analysis.

  6. 21 CFR 864.7040 - Adenosine triphosphate release assay.


    ... device that measures the release of adenosine triphosphate (ATP) from platelets following aggregation. This measurement is made on platelet-rich plasma using a photometer and a luminescent firefly extract. Simultaneous measurements of platelet aggregation and ATP release are used to evaluate platelet...

  7. No role of interstitial adenosine in insulin-mediated vasodilation

    Dela, F; Stallknecht, B


    The mechanisms behind the vasodilatory effect of insulin are not fully understood, but nitric oxide plays an important role. We have investigated the possibility that insulin mediates vasodilatation in the human skeletal muscle via an increase in extracellular adenosine concentrations. In eight h...

  8. Intracortical injection of endothelin-1 induces cortical infarcts in mice: effect of neuronal expression of an adenosine transporter

    Soylu Hanifi


    Full Text Available Abstract Background Activation of adenosine A1 receptors has neuroprotective effects in animal stroke models. Adenosine levels are regulated by nucleoside transporters. In vitro studies showed that neuron-specific expression of human equilibrative nucleoside transporter 1 (hENT1 decreases extracellular adenosine levels and adenosine A1 receptor activity. In this study, we tested the effect of hENT1 expression on cortical infarct size following intracerebral injection of the vasoconstrictor endothelin-1 (ET-1 or saline. Methods Mice underwent stereotaxic intracortical injection of ET-1 (1 μl; 400 pmol or saline (1 μl. Some mice received the adenosine receptor antagonist caffeine (25 mg/kg, intraperitoneal 30 minutes prior to ET-1. Perfusion and T2-weighted magnetic resonance imaging (MRI were used to measure cerebral blood flow (CBF and subsequent infarct size, respectively. Results ET-1 reduced CBF at the injection site to 7.3 ± 1.3% (n = 12 in hENT1 transgenic (Tg and 12.5 ± 2.0% (n = 13 in wild type (Wt mice. At 48 hours following ET-1 injection, CBF was partially restored to 35.8 ± 4.5% in Tg and to 45.2 ± 6.3% in Wt mice; infarct sizes were significantly greater in Tg (9 ± 1.1 mm3 than Wt (5.4 ± 0.8 mm3 mice. Saline-treated Tg and Wt mice had modest decreases in CBF and infarcts were less than 1 mm3. For mice treated with caffeine, CBF values and infarct sizes were not significantly different between Tg and Wt mice. Conclusions ET-1 produced greater ischemic injury in hENT1 Tg than in Wt mice. This genotype difference was not observed in mice that had received caffeine. These data indicate that hENT1 Tg mice have reduced ischemia-evoked increases in adenosine receptor activity compared to Wt mice.

  9. Facile synthesis of N-6 adenosine modified analogue toward S-adenosyl methionine derived probe for protein arginine methyltransferases

    Wei Hong; James Dowden


    Chemically modified cellular co-factors that provide function, such as immobilization or incorporation of fluorescent dyes, are valuable probes of biological activity. A convenient route to obtain S-adenosyl methionine (AdoMet) analogues modified at N-6 adenosine to feature a linker terminating in azide functionality is described herein. Subsequent decoration of such AdoMet analogues with guanidinium terminated linkers leads to novel potential bisubstrate inhibitors for protein arginine methyltransferases, PRMTs.

  10. AMPKα2基因克隆及其野生型和突变型真核表达载体的构建%Cloning of activating adenosine monophosphate-activated protein kinase alpha 2 subunit gene and construction of its wild-type and mutant eukaryotic expression vectors

    罗招凡; 李芳萍; 丁鹤林; 程桦


    背景:实验表明,通过激活单磷酸腺苷激活的蛋白激酶(AMP-Activated Protein Kinase, AMPK)α2可以增加胰岛素的敏感性和骨骼肌葡萄糖的摄取,其有望成为预防和治疗2型糖尿病的新的生理和药理作用靶点.目的:克隆人的AMPKα2基因,并构建其野生型和突变型真核表达载体.设计:单一样本观察.时间及地点:实验于2007-04/2008-01在中山大学附属第二医院临床分子生物实验室完成.材料:QuikChange Ⅱ Site-Directed Mutagenesis Kit为Stratagene公司产品.真核表达载体pcDNA3.1(+),大肠杆菌DH5α为实验室保存.人骨胳肌组织来源于中山大学附属第二医院手术截肢患者,获患者知情同意,新鲜取材后液氮冷冻.方法:采用反转录一聚合酶链反应技术从人骨骼肌扩增AMPKα2基因,并将其克隆到T载体,通过测序对其进行鉴定.采用Quickchange定点诱变试剂盒对AMPKα2基因进行体外定点诱变,并将其野生和突变的编码基因亚克隆到真核表达载体pcDNA3.1中,通过酶切和测序进行鉴定.主要观察指标:①目的基因的克隆.②定点诱变.③真核表达质粒的构建.结果:成功克隆了AMPKα2基因,大小约1 700 bp,与已发表的AMPKα2同源性为99%,GenBank录入号EF056019.成功将AMPKα2第45位Lysine(AAA)突变为Arginine(AGA),成功构建了野生型和突变型pcDNA-AMPK α2重组质粒.结论:实验成功克隆了AMPKα2基因,构建了其野生型和突变型真核表达载体.%BACKGROUND: The experimental results showed that insulin sensitivity and glucose uptake in skeletal muscle could be improved by activating adenosine monophosphate-activated protein kinase a2 (AMPKα2). AMPKa2 is expected to become a new physiological and pharmacological target for the prevention and treatment of type 2 diabetes mellitus. OBJECTIVE: To clone human AMPKa2 subunit gene and to construct its wild-type and mutant eukaryotic expression vectors. DESIGN: A single sample observation

  11. Stimulation of NTS A1 adenosine receptors differentially resets baroreflex control of regional sympathetic outputs.

    Scislo, Tadeusz J; Ichinose, Tomoko K; O'Leary, Donal S


    Previously we showed that pressor and differential regional sympathoexcitatory responses (adrenal > renal >/= lumbar) evoked by stimulation of A(1) adenosine receptors located in the nucleus of the solitary tract (NTS) were attenuated/abolished by baroreceptor denervation or blockade of glutamatergic transmission in the NTS, suggesting A(1) receptor-elicited inhibition of glutamatergic transmission in baroreflex pathways. Therefore we tested the hypothesis that stimulation of NTS A(1) adenosine receptors differentially inhibits/resets baroreflex responses of preganglionic adrenal (pre-ASNA), renal (RSNA), and lumbar (LSNA) sympathetic nerve activity. In urethane-chloralose-anesthetized male Sprague-Dawley rats (n = 65) we compared baroreflex-response curves (iv nitroprusside and phenylephrine) evoked before and after bilateral microinjections into the NTS of A(1) adenosine receptor agonist (N(6)-cyclopentyladenosine, CPA; 0.033-330 pmol/50 nl). CPA evoked typical dose-dependent pressor and differential sympathoexcitatory responses and similarly shifted baroreflex curves for pre-ASNA, RSNA, and LSNA toward higher mean arterial pressure (MAP) in a dose-dependent manner; the maximal shifts were 52.6 +/- 2.8, 48.0 +/- 3.6, and 56.8 +/- 6.7 mmHg for pre-ASNA, RSNA, and LSNA, respectively. These shifts were not a result of simple baroreceptor resetting because they were two to three times greater than respective increases in baseline MAP evoked by CPA. Baroreflex curves for pre-ASNA were additionally shifted upward: the maximal increases of upper and lower plateaus were 41.8 +/- 16.4% and 45.3 +/- 8.7%, respectively. Maximal gain (%/mmHg) measured before vs. after CPA increased for pre-ASNA (3.0 +/- 0.6 vs. 4.9 +/- 1.3), decreased for RSNA (4.1 +/- 0.6 vs. 2.3 +/- 0.3), and remained unaltered for LSNA (2.1 +/- 0.2 vs. 2.0 +/- 0.1). Vehicle control did not alter the baroreflex curves. We conclude that the activation of NTS A(1) adenosine receptors differentially inhibits

  12. Topological sub-structural molecular design (TOPS-MODE): a useful tool to explore key fragments of human A3 adenosine receptor ligands.

    Saíz-Urra, Liane; Teijeira, Marta; Rivero-Buceta, Virginia; Helguera, Aliuska Morales; Celeiro, Maria; Terán, Ma Carmen; Besada, Pedro; Borges, Fernanda


    Adenosine regulates tissue function by activating four G-protein-coupled adenosine receptors (ARs). Selective agonists and antagonists for A3 ARs have been investigated for the treatment of a variety of immune disorders, cancer, brain, and heart ischemic conditions. We herein present a QSAR study based on a Topological sub-structural molecular design (TOPS-MODE) approach, intended to predict the A3 ARs of a diverse dataset of 124 (94 training set/ 30 prediction set) adenosine derivatives. The final model showed good fit and predictive capability, displaying 85.1 % of the experimental variance. The TOPS-MODE approach afforded a better understanding and interpretation of the developed model based on the useful information extracted from the analysis of the contribution of different molecular fragments to the affinity.

  13. Intracerebral adenosine infusion improves neurological outcome after transient focal ischemia in rats.

    Kitagawa, Hisashi; Mori, Atsushi; Shimada, Jun; Mitsumoto, Yasuhide; Kikuchi, Tetsuro


    Second Institute of New Drug Research, Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan In order to elucidate the role of adenosine in brain ischemia, the possible protective effects of adenosine on ischemic brain injury were investigated in a rat model of brain ischemia both in vitro and in vivo. Exogenous adenosine dose-dependently rescued cortical neuronal cells from injury after glucose deprivation in vitro. Adenosine (1 mM) also significantly reduced hypoglycemia/hypoxia-induced glutamate release from the hippocampal slice. In a rat model of transient middle cerebral artery occlusion (MCAO), extracellular adenosine concentration was increased immediately after occlusion, and then returned to the baseline by 30 min after reperfusion. Adenosine infusion through a microdialysis probe into the ipsilateral striatum (1 mM adenosine, 2 microl min(-1), total 4.5 h from the occlusion to 3 h after reperfusion) showed a significant improvement in the neurological outcome, and about 25% reduction of infarct volume, although the effect did not reach statistical significance, compared with the vehicle-treated group at 20 h after 90 min of MCAO. These results demonstrated the neuroprotective effect of adenosine against ischemic brain injury both in vitro and in vivo, suggesting the possible therapeutic application of adenosine regulating agents, which inhibit adenosine uptake or metabolism to enhance or maintain extracellular endogenous adenosine levels, for stroke treatment.

  14. Adenosine A2B receptor-mediated leukemia inhibitory factor release from astrocytes protects cortical neurons against excitotoxicity

    Moidunny Shamsudheen


    Full Text Available Abstract Background Neuroprotective and neurotrophic properties of leukemia inhibitory factor (LIF have been widely reported. In the central nervous system (CNS, astrocytes are the major source for LIF, expression of which is enhanced following disturbances leading to neuronal damage. How astrocytic LIF expression is regulated, however, has remained an unanswered question. Since neuronal stress is associated with production of extracellular adenosine, we investigated whether LIF expression in astrocytes was mediated through adenosine receptor signaling. Methods Mouse cortical neuronal and astrocyte cultures from wild-type and adenosine A2B receptor knock-out animals, as well as adenosine receptor agonists/antagonists and various enzymatic inhibitors, were used to study LIF expression and release in astrocytes. When needed, a one-way analysis of variance (ANOVA followed by Bonferroni post-hoc test was used for statistical analysis. Results We show here that glutamate-stressed cortical neurons induce LIF expression through activation of adenosine A2B receptor subtype in cultured astrocytes and require signaling of protein kinase C (PKC, mitogen-activated protein kinases (MAPKs: p38 and ERK1/2, and the nuclear transcription factor (NF-κB. Moreover, LIF concentration in the supernatant in response to 5′-N-ethylcarboxamide (NECA stimulation was directly correlated to de novo protein synthesis, suggesting that LIF release did not occur through a regulated release pathway. Immunocytochemistry experiments show that LIF-containing vesicles co-localize with clathrin and Rab11, but not with pHogrin, Chromogranin (CgA and CgB, suggesting that LIF might be secreted through recycling endosomes. We further show that pre-treatment with supernatants from NECA-treated astrocytes increased survival of cultured cortical neurons against glutamate, which was absent when the supernatants were pre-treated with an anti-LIF neutralizing antibody. Conclusions

  15. Adenosine-A1 receptor agonist induced hyperalgesic priming type II.

    Araldi, Dioneia; Ferrari, Luiz F; Levine, Jon D


    We have recently shown that repeated exposure of the peripheral terminal of the primary afferent nociceptor to the mu-opioid receptor (MOR) agonist DAMGO ([D-Ala, N-Me-Phe, Gly-ol]-enkephalin acetate salt) induces a model of transition to chronic pain that we have termed type II hyperalgesic priming. Similar to type I hyperalgesic priming, there is a markedly prolonged response to subsequent administration of proalgesic cytokines, prototypically prostaglandin E2 (PGE2). However, type II hyperalgesic priming differs from type I in being rapidly induced, protein kinase A (PKA), rather than PKCε dependent, not reversed by a protein translation inhibitor, occurring in female as well as in male rats, and isolectin B4-negative neuron dependent. We report that, as with the repeated injection of a MOR agonist, the repeated administration of an agonist at the A1-adenosine receptor, also a Gi-protein coupled receptor, N-cyclopentyladenosine (CPA), also produces priming similar to DAMGO-induced type II hyperalgesic priming. In this study, we demonstrate that priming induced by repeated exposure to this A1-adenosine receptor agonist shares the same mechanisms, as MOR-agonist induced priming. However, the prolongation of PGE2 hyperalgesia induced by repeated administration of CPA depends on G-protein αi subunit activation, differently from DAMGO-induced type II priming, in which it depends on the β/γ subunit. These data implicate a novel form of Gi-protein signaling pathway in the type II hyperalgesic priming induced by repeated administration of an agonist at A1-adenosine receptor to the peripheral terminal of the nociceptor.

  16. Adenosine transiently modulates stimulated dopamine release in the caudate-putamen via A1 receptors.

    Ross, Ashley E; Venton, B Jill


    Adenosine modulates dopamine in the brain via A1 and A2A receptors, but that modulation has only been characterized on a slow time scale. Recent studies have characterized a rapid signaling mode of adenosine that suggests a possible rapid modulatory role. Here, fast-scan cyclic voltammetry was used to characterize the extent to which transient adenosine changes modulate stimulated dopamine release (5 pulses at 60 Hz) in rat caudate-putamen brain slices. Exogenous adenosine was applied and dopamine concentration monitored. Adenosine only modulated dopamine when it was applied 2 or 5 s before stimulation. Longer time intervals and bath application of 5 μM adenosine did not decrease dopamine release. Mechanical stimulation of endogenous adenosine 2 s before dopamine stimulation also decreased stimulated dopamine release by 41 ± 7%, similar to the 54 ± 6% decrease in dopamine after exogenous adenosine application. Dopamine inhibition by transient adenosine was recovered within 10 min. The A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine blocked the dopamine modulation, whereas dopamine modulation was unaffected by the A2A receptor antagonist SCH 442416. Thus, transient adenosine changes can transiently modulate phasic dopamine release via A1 receptors. These data demonstrate that adenosine has a rapid, but transient, modulatory role in the brain. Here, transient adenosine was shown to modulate phasic dopamine release on the order of seconds by acting at the A1 receptor. However, sustained increases in adenosine did not regulate phasic dopamine release. This study demonstrates for the first time a transient, neuromodulatory function of rapid adenosine to regulate rapid neurotransmitter release.

  17. Adenosine A(2A) receptor modulation of hippocampal CA3-CA1 synapse plasticity during associative learning in behaving mice.

    Fontinha, Bruno M; Delgado-García, José M; Madroñal, Noelia; Ribeiro, Joaquim A; Sebastião, Ana M; Gruart, Agnès


    Previous in vitro studies have characterized the electrophysiological and molecular signaling pathways of adenosine tonic modulation on long-lasting synaptic plasticity events, particularly for hippocampal long-term potentiation (LTP). However, it remains to be elucidated whether the long-term changes produced by endogenous adenosine in the efficiency of synapses are related to those required for learning and memory formation. Our goal was to understand how endogenous activation of adenosine excitatory A(2A) receptors modulates the associative learning evolution in conscious behaving mice. We have studied here the effects of the application of a highly selective A(2A) receptor antagonist, SCH58261, upon a well-known associative learning paradigm-classical eyeblink conditioning. We used a trace paradigm, with a tone as the conditioned stimulus (CS) and an electric shock presented to the supraorbital nerve as the unconditioned stimulus (US). A single electrical pulse was presented to the Schaffer collateral-commissural pathway to evoke field EPSPs (fEPSPs) in the pyramidal CA1 area during the CS-US interval. In vehicle-injected animals, there was a progressive increase in the percentage of conditioning responses (CRs) and in the slope of fEPSPs through conditioning sessions, an effect that was completely prevented (and lost) in SCH58261 (0.5 mg/kg, i.p.) -injected animals. Moreover, experimentally evoked LTP was impaired in SCH58261-injected mice. In conclusion, the endogenous activation of adenosine A(2A) receptors plays a pivotal effect on the associative learning process and its relevant hippocampal circuits, including activity-dependent changes at the CA3-CA1 synapse.

  18. AMP-guided tumour-specific nanoparticle delivery via adenosine A1 receptor.

    Dai, Tongcheng; Li, Na; Han, Fajun; Zhang, Hua; Zhang, Yuanxing; Liu, Qin


    Active targeting-ligands have been increasingly used to functionalize nanoparticles for tumour-specific clinical applications. Here we utilize nucleotide adenosine 5'-monophosphate (AMP) as a novel ligand to functionalize polymer-based fluorescent nanoparticles (NPs) for tumour-targeted imaging. We demonstrate that AMP-conjugated NPs (NPs-AMP) efficiently bind to and are following internalized into colon cancer cell CW-2 and breast cancer cell MDA-MB-468 in vitro. RNA interference and inhibitor assays reveal that the targeting effects mainly rely on the specific binding of AMP to adenosine A1 receptor (A1R), which is greatly up-regulated in cancer cells than in matched normal cells. More importantly, NPs-AMP specifically accumulate in the tumour site of colon and breast tumour xenografts and are further internalized into the tumour cells in vivo via tail vein injection, confirming that the high in vitro specificity of AMP can be successfully translated into the in vivo efficacy. Furthermore, NPs-AMP exhibit an active tumour-targeting behaviour in various colon and breast cancer cells, which is positively related to the up-regulation level of A1R in cancer cells, suggesting that AMP potentially suits for more extensive A1R-overexpressing cancer models. This work establishes AMP to be a novel tumour-targeting ligand and provides a promising strategy for future diagnostic or therapeutic applications.

  19. Moderate exercise training promotes adaptations in coronary blood flow and adenosine production in normotensive rats

    Fernanda R. Roque


    Full Text Available OBJECTIVES: Aerobic exercise training prevents cardiovascular risks. Regular exercise promotes functional and structural adaptations that are associated with several cardiovascular benefits. The aim of this study is to investigate the effects of swimming training on coronary blood flow, adenosine production and cardiac capillaries in normotensive rats. METHODS: Wistar rats were randomly divided into two groups: control (C and trained (T. An exercise protocol was performed for 10 weeks and 60 min/day with a tail overload of 5% bodyweight. Coronary blood flow was quantified with a color microsphere technique, and cardiac capillaries were quantified using light microscopy. Adenine nucleotide hydrolysis was evaluated by enzymatic activity, and protein expression was evaluated by western blot. The results are presented as the means ± SEMs (p<0.05. RESULTS: Exercise training increased the coronary blood flow and the myocardial capillary-to-fiber ratio. Moreover, the circulating and cardiac extracellular adenine nucleotide hydrolysis was higher in the trained rats than in the sedentary rats due to the increased activity and protein expression of enzymes, such as E-NTPDase and 59- nucleotidase. CONCLUSIONS: Swimming training increases coronary blood flow, number of cardiac capillaries, and adenine nucleotide hydrolysis. Increased adenosine production may be an important contributor to the enhanced coronary blood flow and angiogenesis that were observed in the exercise-trained rats; collectively, these results suggest improved myocardial perfusion.

  20. CD73-derived adenosine and tenascin-C control cytokine production by epicardium-derived cells formed after myocardial infarction.

    Hesse, Julia; Leberling, Stella; Boden, Elisabeth; Friebe, Daniela; Schmidt, Timo; Ding, Zhaoping; Dieterich, Peter; Deussen, Andreas; Roderigo, Claudia; Rose, Christine R; Floss, Doreen M; Scheller, Jürgen; Schrader, Jürgen


    Epicardium-derived cells (EPDCs) play a fundamental role in embryonic cardiac development and are reactivated in the adult heart in response to myocardial infarction (MI). In this study, EPDCs from post-MI rat hearts highly expressed the ectoenzyme CD73 and secreted the profibrotic matricellular protein tenascin-C (TNC). CD73 on EPDCs extensively generated adenosine from both extracellular ATP and NAD. This in turn stimulated the release of additional nucleotides from a Brefeldin A-sensitive intracellular pool via adenosine-A2BR signaling, forming a positive-feedback loop. A2BR activation in addition strongly promoted the release of major regulatory cytokines, such as IL-6, IL-11, and VEGF. TNC was found to stimulate EPDC migration and, together with ATP-P2X7R signaling, to activate inflammasomes in EPDCs via TLR4. Our results demonstrate that EPDCs are an important source of various proinflammatory factors in the post-MI heart controlled by purinergic and TNC signaling.-Hesse, J., Leberling, S., Boden, E., Friebe, D., Schmidt, T., Ding, Z., Dieterich, P., Deussen, A., Roderigo, C., Rose, C. R., Floss, D. M., Scheller, J., Schrader, J. CD73-derived adenosine and tenascin-C control cytokine production by epicardium-derived cells formed after myocardial infarction.

  1. An adenosine A(2A) antagonist injected in the NTS reverses thermal prolongation of the LCR in decerebrate piglets.

    Xia, Luxi; Bartlett, Donald; Leiter, J C


    Hyperthermia prolongs the laryngeal chemoreflex (LCR). Under normothermic conditions, adenosine antagonists shorten and adenosine A(2A) (Ad-A(2A)) agonists prolong the LCR. Therefore, we tested the hypothesis that SCH-58261, an Ad-A(2A) receptor antagonist, would prevent thermal prolongation of the LCR when injected unilaterally within the nucleus of the solitary tract (NTS). We studied decerebrate piglets aged 4-13 days. We elicited the LCR by injecting 0.1ml of water into the larynx and recorded integrated phrenic nerve activity. The laryngeal chemoreflex was prolonged when the body temperature of each piglet was raised approximately 2.5 degrees C, and SCH-58261 reversed the thermal prolongation of the LCR when injected into the NTS (n=13), but not when injected in the nucleus ambiguus (n=9). Injections of vehicle alone into the NTS did not alter the thermal prolongation of the LCR (n=9). We conclude that activation of adenosine receptors, perhaps located on GABAergic neurons in the NTS, contributes to thermal prolongation of the LCR.

  2. Modulation of synaptic transmission by adenosine in layer 2/3 of the rat visual cortex in vitro.

    Bannon, N M; Zhang, P; Ilin, V; Chistiakova, M; Volgushev, M


    Adenosine is a wide-spread endogenous neuromodulator. In the central nervous system it activates A1 and A2A receptors (A1Rs and A2ARs) which have differential distributions, different affinities to adenosine, are coupled to different G-proteins, and have opposite effects on synaptic transmission. Although effects of adenosine are studied in detail in several brain areas, such as the hippocampus and striatum, the heterogeneity of the effects of A1R and A2AR activation and their differential distribution preclude generalization over brain areas and cell types. Here we study adenosine's effects on excitatory synaptic transmission to layer 2/3 pyramidal neurons in slices of the rat visual cortex. We measured effects of bath application of adenosine receptor ligands on evoked excitatory postsynaptic potentials (EPSPs), miniature excitatory postsynaptic potentials (mEPSPs), and membrane properties. Adenosine reduced the amplitude of evoked EPSPs and excitatory postsynaptic currents (EPSCs), and reduced frequency of mEPSPs in a concentration-dependent and reversible manner. Concurrent with EPSP/C amplitude reduction was an increase in the paired-pulse ratio. These effects were blocked by application of the selective A1R antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine), suggesting that activation of presynaptic A1Rs suppresses excitatory transmission by reducing release probability. Adenosine (20μM) hyperpolarized the cell membrane from -65.3±1.5 to -67.7±1.8mV, and reduced input resistance from 396.5±44.4 to 314.0±36.3MOhm (∼20%). These effects were also abolished by DPCPX, suggesting postsynaptic A1Rs. Application of the selective A2AR antagonist SCH-58261 (2-(2-furanyl)-7-(2-phenylethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-a-mine) on the background of high adenosine concentrations revealed an additional decrease in EPSP amplitude. Moreover, application of the A2AR agonist CGS-21680 (4-[2-[[6-amino-9-(N-ethyl-β-d-ribofuranuronamidosyl)-9H

  3. Genetics and complementation of Haemophilus influenzae mutants deficient in adenosine 5'-triphosphate-dependent nuclease

    Kooistra, J.; Small, G.D.; Setlow, J.K.; Shapanka, R.


    Eight different mutations in Haemophilus influenzae leading to deficiency in adenosine 5'-triphosphate (ATP)-dependent nuclease have been investigated in strains in which the mutations of the originally mutagenized strains have been transferred into the wild type. Sensitivity to mitomycin C and deoxycholate and complementation between extracts and deoxyribonucleic acid (DNA)-dependent ATPase activity have been measured. Genetic crosses have provided information on the relative position of the mutations on the genome. There are three complementation groups, corresponding to three genetic groups. The strains most sensitive to mitomycin and deoxycholate, derived from mutants originally selected on the basis of sensitivity to mitomycin C or methyl methanesulfonate, are in one group. Apparently all these sensitive strains lack DNA-dependent ATPase activity, as does a strain intermediate in sensitivity to deoxycholate, which is the sole representative of another group. There are four strains that are relatively resistant to deoxycholate and mitomycin C, and all of these contain the ATPase activity.

  4. Expression of human adenosine deaminase in murine hematopoietic cells.

    Belmont, J W; MacGregor, G R; Wager-Smith, K; Fletcher, F A; Moore, K A; Hawkins, D; Villalon, D; Chang, S M; Caskey, C T


    Multiple replication-defective retrovirus vectors were tested for their ability to transfer and express human adenosine deaminase in vitro and in vivo in a mouse bone marrow transplantation model. High-titer virus production was obtained from vectors by using both a retrovirus long terminal repeat promoter and internal transcriptional units with human c-fos and herpes virus thymidine kinase promoters. After infection of primary murine bone marrow with one of these vectors, human adenosine deaminase was detected in 60 to 85% of spleen colony-forming units and in the blood of 14 of 14 syngeneic marrow transplant recipients. This system offers the opportunity to assess methods for increasing efficiency of gene transfer, for regulation of expression of foreign genes in hematopoietic progenitors, and for long-term measurement of the stability of expression in these cells. Images PMID:3072474

  5. Evidence for an A1-adenosine receptor in the guinea-pig atrium.

    Collis, M. G.


    1 The purpose of this study was to determine whether the adenosine receptor that mediates a decrease in the force of contraction of the guinea-pig atrium is of the A1- or A2-sub-type. 2 Concentration-response curves to adenosine and a number of 5'- and N6-substituted analogues were constructed and the order of potency of the purines was: 5'-N-cyclopropylcarboxamide adenosine (NCPCA) = 5'-N-ethylcarboxamide adenosine (NECA) greater than N6cyclohexyladenosine (CHA) greater than L-N6-phenylisopropyl adenosine (L-PIA) = 2-chloroadenosine- greater than adenosine greater than D-N6-phenylisopropyl adenosine (D-PIA). 3 The difference in potency between the stereoisomers D- and L-PIA was over 100 fold. 4 The adenosine transport inhibitor, dipyridamole, potentiated submaximal responses to adenosine but had no significant effect on those evoked by the other purines. 5 Theophylline antagonized responses evoked by all purines, and with D-PIA revealed a positive inotropic effect that was abolished by atenolol. 6 The results indicate the existence of an adenosine A1-receptor in the guinea-pig atrium. PMID:6297647

  6. Adenosine-mediated modulation of ventral horn interneurons and spinal motoneurons in neonatal mice.

    Witts, Emily C; Nascimento, Filipe; Miles, Gareth B


    Neuromodulation allows neural networks to adapt to varying environmental and biomechanical demands. Purinergic signaling is known to be an important modulatory system in many parts of the CNS, including motor control circuitry. We have recently shown that adenosine modulates the output of mammalian spinal locomotor control circuitry (Witts EC, Panetta KM, Miles GB. J Neurophysiol 107: 1925-1934, 2012). Here we investigated the cellular mechanisms underlying this adenosine-mediated modulation. Whole cell patch-clamp recordings were performed on ventral horn interneurons and motoneurons within in vitro mouse spinal cord slice preparations. We found that adenosine hyperpolarized interneurons and reduced the frequency and amplitude of synaptic inputs to interneurons. Both effects were blocked by the A1-type adenosine receptor antagonist DPCPX. Analysis of miniature postsynaptic currents recorded from interneurons revealed that adenosine reduced their frequency but not amplitude, suggesting that adenosine acts on presynaptic receptors to modulate synaptic transmission. In contrast to interneurons, recordings from motoneurons revealed an adenosine-mediated depolarization. The frequency and amplitude of synaptic inputs to motoneurons were again reduced by adenosine, but we saw no effect on miniature postsynaptic currents. Again these effects on motoneurons were blocked by DPCPX. Taken together, these results demonstrate differential effects of adenosine, acting via A1 receptors, in the mouse spinal cord. Adenosine has a general inhibitory action on ventral horn interneurons while potentially maintaining motoneuron excitability. This may allow for adaptation of the locomotor pattern generated by interneuronal networks while helping to ensure the maintenance of overall motor output.

  7. Adenosine in the tuberomammillary nucleus inhibits the histaminergic system via A1 receptors and promotes non-rapid eye movement sleep.

    Oishi, Yo; Huang, Zhi-Li; Fredholm, Bertil B; Urade, Yoshihiro; Hayaishi, Osamu


    Adenosine has been proposed to promote sleep through A(1) receptors (A(1)R's) and/or A(2A) receptors in the brain. We previously reported that A(2A) receptors mediate the sleep-promoting effect of prostaglandin D(2), an endogenous sleep-inducing substance, and that activation of these receptors induces sleep and blockade of them by caffeine results in wakefulness. On the other hand, A(1)R has been suggested to increase sleep by inhibition of the cholinergic region of the basal forebrain. However, the role and target sites of A(1)R in sleep-wake regulation remained controversial. In this study, immunohistochemistry revealed that A(1)R was expressed in histaminergic neurons of the rat tuberomammillary nucleus (TMN). In vivo microdialysis showed that the histamine release in the frontal cortex was decreased by microinjection into the TMN of N(6)-cyclopentyladenosine (CPA), an A(1)R agonist, adenosine or coformycin, an inhibitor of adenosine deaminase, which catabolizes adenosine to inosine. Bilateral injection of CPA into the rat TMN significantly increased the amount and the delta power density of non-rapid eye movement (non-REM; NREM) sleep but did not affect REM sleep. CPA-promoted sleep was observed in WT mice but not in KO mice for A(1)R or histamine H(1) receptor, indicating that the NREM sleep promoted by A(1)R-specific agonist depended on the histaminergic system. Furthermore, the bilateral injection of adenosine or coformycin into the rat TMN increased NREM sleep, which was completely abolished by coadministration of 1,3-dimethyl-8-cyclopenthylxanthine, a selective A(1)R antagonist. These results indicate that endogenous adenosine in the TMN suppresses the histaminergic system via A(1)R to promote NREM sleep.

  8. Adenosine receptors and stress : Studies using methylmercury, caffeine and hypoxia

    Björklund, Olga


    Brain development is a precisely organized process that can be disturbed by various stress factors present in the diet (e.g. exposure to xenobiotics) as well as insults such as decreased oxygen supply. The consequent adverse changes in nervous system function may not necessarily be apparent until a critical age when neurodevelopmental defects may be unmasked by a subsequent challenge. Adenosine and its receptors (AR) (A1, A2A, A2B and A3) which participate in the brain stres...

  9. Severe combined immunodeficiency due to adenosine deaminase deficiency.

    Hussain, Waqar; Batool, Asma; Ahmed, Tahir Aziz; Bashir, Muhammad Mukarram


    Severe Combined Immunodeficiency is the term applied to a group of rare genetic disorders characterised by defective or absent T and B cell functions. Patients usually present in first 6 months of life with respiratory/gastrointestinal tract infections and failure to thrive. Among the various types of severe combined immunodeficiency, enzyme deficiencies are relatively less common. We report the case of a 6 years old girl having severe combined immunodeficiency due to adenosine deaminase deficiency.

  10. Dosagem sérica de adenosina deaminase em lúpus eritematoso sistêmico: ausência de associação com atividade de doença Levels of serum adenosine deaminase in systemic lupus erythematosus: lack of association with disease activity

    Isabella Lima


    Full Text Available O lúpus eritematoso sistêmico (LES é uma doença inflamatória auto-imune, que evolui intercalando períodos de atividade e remissão. OBJETIVO: avaliar a associação da dosagem sérica de adenosina deaminase (ADA e atividade do LES, segundo os critérios do SLEDAI 2K - Systemic lupus erythematosus disease activity index. MÉTODOS: avaliou-se 82 pacientes com LES atendidos em um hospital de referência para o tratamento do LES em Salvador, BA, Brasil. A atividade de doença foi determinada pelo SLEDAI 2K e a dosagem sérica da ADA realizada por colorimetria. RESULTADOS: oitenta e uma pacientes (98,78% eram do sexo feminino e a idade média foi de 35,07±11,73 anos. O escore de SLEDAI médio foi de 11,66±5,89; a média de ADA sérica foi de 38,24±13,61U/l; C3 de 91,93±27,39 mg/dl; C4 de 15,17±5,77 mg/dl e a pesquisa de anticorpos anti-DNA nativo (aDNAn foi positiva em 31 casos (37,8%. Não houve correlação entre os níveis séricos de ADA e escore do SLEDAI. A ADA sérica correlacionou-se inversamente com C4 (r=-0,336 e p=0,001. CONCLUSÕES: no presente estudo a dosagem sérica de ADA não se associou a atividade de doença segundo os critérios do SLEDAI 2K, sugerindo que esse teste não deve ser utilizado como marcador de atividade de doença em LES. Esse resultado diverge da maioria dos trabalhos publicados, o que pode ser explicado pela dificuldade de padronização da técnica de dosagem da ADA ou por diferença nas diversas populações estudadas.Systemic lupus erythematosus (SLE is an autoimmune inflammatory disease, with a variable course and characterized by periods of remissions and exacerbations. OBJECTIVE: To evaluate the association between serum adenosine deaminase (ADA levels and disease activity in SLE. METHODS: Eighty two SLE patients seen at Santa Izabel Hospital in Salvador, BA, Brazil, were studied. Disease activity was measured by SLEDAI 2K- Systemic Lupus Erythematosus Disease Activity Index, and serum ADA was

  11. The synthesis of a series of adenosine A3 receptor agonists.

    Broadley, Kenneth J; Burnell, Erica; Davies, Robin H; Lee, Alan T L; Snee, Stephen; Thomas, Eric J


    A series of 1'-(6-aminopurin-9-yl)-1'-deoxy-N-methyl-β-d-ribofuranuronamides that were characterised by 2-dialkylamino-7-methyloxazolo[4,5-b]pyridin-5-ylmethyl substituents on N6 of interest for screening as selective adenosine A3 receptor agonists, have been synthesised. This work involved the synthesis of 2-dialkylamino-5-aminomethyl-7-methyloxazolo[4,5-b]pyridines and analogues that were coupled with the known 1'-(6-chloropurin-9-yl)-1'-deoxy-N-methyl-β-d-ribofuranuronamide. The oxazolo[4,5-b]pyridines were synthesized by regioselective functionalisation of 2,4-dimethylpyridine N-oxides. The regioselectivities of these reactions were found to depend upon the nature of the heterocycle with 2-dimethylamino-5,7-dimethyloxazolo[4,5-b]pyridine-N-oxide undergoing regioselective functionalisation at the 7-methyl group on reaction with trifluoroacetic anhydride in contrast to the reaction of 4,6-dimethyl-3-hydroxypyridine-N-oxide with acetic anhydride that resulted in functionalisation of the 6-methyl group. To optimise selectivity for the A3 receptor, 5-aminomethyl-7-bromo-2-dimethylamino-4-[(3-methylisoxazol-5-yl)methoxy]benzo[d]oxazole was synthesised and coupled with the 1'-(6-chloropurin-9-yl)-1'-deoxy-N-methyl-β-d-ribofuranuronamide. The products were active as selective adenosine A3 agonists.

  12. Adenosine receptors in post-mortem human brain.

    James, S; Xuereb, J H; Askalan, R; Richardson, P J


    1. Adenosine A2-like binding sites were characterized in post-mortem human brain membranes by examining several compounds for their ability to displace [3H]-CGS 21680 (2[p-(2 carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamido adenosine) binding. 2. Two A2-like binding sites were identified in the striatum. 3. The more abundant striatal site was similar to the A2a receptor previously described in rat striatum, both in its pharmacological profile and striatal localization. 4. The less abundant striatal site had a pharmacological profile similar to that of the binding site characterized in the other brain regions examined. This was intermediate in character between A1 and A2 and may represent another adenosine receptor subtype. 5. The co-purification of [3H]-CGS 21680 binding during immunoisolation of human striatal cholinergic membranes was used to assess the possible cholinergic localization of A2-like binding sites in the human striatum. Only the more abundant striatal site co-purified with cholinergic membranes. This suggests that this A2a-like site is present on cholinergic neurones in the human striatum.

  13. Alterations in the brain adenosine metabolism cause behavioral and neurological impairment in ADA-deficient mice and patients

    Sauer, Aisha V.; Hernandez, Raisa Jofra; Fumagalli, Francesca; Bianchi, Veronica; Poliani, Pietro L.; Dallatomasina, Chiara; Riboni, Elisa; Politi, Letterio S.; Tabucchi, Antonella; Carlucci, Filippo; Casiraghi, Miriam; Carriglio, Nicola; Cominelli, Manuela; Forcellini, Carlo Alberto; Barzaghi, Federica; Ferrua, Francesca; Minicucci, Fabio; Medaglini, Stefania; Leocani, Letizia; la Marca, Giancarlo; Notarangelo, Lucia D.; Azzari, Chiara; Comi, Giancarlo; Baldoli, Cristina; Canale, Sabrina; Sessa, Maria; D’Adamo, Patrizia; Aiuti, Alessandro


    Adenosine Deaminase (ADA) deficiency is an autosomal recessive variant of severe combined immunodeficiency (SCID) caused by systemic accumulation of ADA substrates. Neurological and behavioral abnormalities observed in ADA-SCID patients surviving after stem cell transplantation or gene therapy represent an unresolved enigma in the field. We found significant neurological and cognitive alterations in untreated ADA-SCID patients as well as in two groups of patients after short- and long-term enzyme replacement therapy with PEG-ADA. These included motor dysfunction, EEG alterations, sensorineural hypoacusia, white matter and ventricular alterations in MRI as well as a low mental development index or IQ. Ada-deficient mice were significantly less active and showed anxiety-like behavior. Molecular and metabolic analyses showed that this phenotype coincides with metabolic alterations and aberrant adenosine receptor signaling. PEG-ADA treatment corrected metabolic adenosine-based alterations, but not cellular and signaling defects, indicating an intrinsic nature of the neurological and behavioral phenotype in ADA deficiency. PMID:28074903

  14. Adenosine A2A receptors and uric acid mediate protective effects of inosine against TNBS-induced colitis in rats.

    Rahimian, Reza; Fakhfouri, Gohar; Daneshmand, Ali; Mohammadi, Hamed; Bahremand, Arash; Rasouli, Mohammad Reza; Mousavizadeh, Kazem; Dehpour, Ahmad Reza


    Inflammatory bowel disease comprises chronic recurrent inflammation of gastrointestinal tract. This study was conducted to investigate inosine, a potent immunomodulator, in 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced chronic model of experimental colitis, and contribution of adenosine A(2A) receptors and the metabolite uric acid as possible underlying mechanisms. Experimental colitis was rendered in rats by a single colonic administration of 10 mg of TNBS. Inosine, potassium oxonate (a hepatic uricase inhibitor), SCH-442416 (a selective adenosine A(2A) receptor antagonist), inosine+potassium oxonate, or inosine+SCH-442416 were given twice daily for 7 successive days. At the end of experiment, macroscopic and histopathologic scores, colonic malondialdehyde (MDA), Tumor Necrosis Factor-alpha (TNF-α) and Interleukin-1beta (IL-1β) levels, and myeloperoxidase (MPO) activity were assessed. Plasma uric acid level was measured throughout the experiment. Both macroscopic and histological features of colonic injury were markedly ameliorated by either inosine, oxonate or inosine+oxonate. Likewise, the elevated amounts of MPO and MDA abated as well as those of TNF-α and IL-1β (Pacid levels were significantly higher in inosine or oxonate groups compared to control. Inosine+oxonate resulted in an even more elvelated uric acid level than each treatment alone (Pacid and adenosine A(2A) receptors contribute to these salutary properties.

  15. Glutamate-induced depression of EPSP-spike coupling in rat hippocampal CA1 neurons and modulation by adenosine receptors.

    Ferguson, Alexandra L; Stone, Trevor W


    The presence of high concentrations of glutamate in the extracellular fluid following brain trauma or ischaemia may contribute substantially to subsequent impairments of neuronal function. In this study, glutamate was applied to hippocampal slices for several minutes, producing over-depolarization, which was reflected in an initial loss of evoked population potential size in the CA1 region. Orthodromic population spikes recovered only partially over the following 60 min, whereas antidromic spikes and excitatory postsynaptic potentials (EPSPs) showed greater recovery, implying a change in EPSP-spike coupling (E-S coupling), which was confirmed by intracellular recording from CA1 pyramidal cells. The recovery of EPSPs was enhanced further by dizocilpine, suggesting that the long-lasting glutamate-induced change in E-S coupling involves NMDA receptors. This was supported by experiments showing that when isolated NMDA-receptor-mediated EPSPs were studied in isolation, there was only partial recovery following glutamate, unlike the composite EPSPs. The recovery of orthodromic population spikes and NMDA-receptor-mediated EPSPs following glutamate was enhanced by the adenosine A1 receptor blocker DPCPX, the A2A receptor antagonist SCH58261 or adenosine deaminase, associated with a loss of restoration to normal of the glutamate-induced E-S depression. The results indicate that the long-lasting depression of neuronal excitability following recovery from glutamate is associated with a depression of E-S coupling. This effect is partly dependent on activation of NMDA receptors, which modify adenosine release or the sensitivity of adenosine receptors. The results may have implications for the use of A1 and A2A receptor ligands as cognitive enhancers or neuroprotectants.

  16. Low-temperature conditioning alleviates chilling injury in loquat fruit and regulates glycine betaine content and energy status.

    Jin, Peng; Zhang, Yu; Shan, Timin; Huang, Yuping; Xu, Jia; Zheng, Yonghua


    The influence of low-temperature conditioning (LTC) treatment on chilling injury, glycine betaine content, and energy metabolism in loquat fruit at 1 °C storage was investigated. The results indicated that LTC treatment significantly reduced chilling injury index, ion leakage, and malondialdehyde content in loquat fruit. Betaine aldehyde hydrogenase (BADH) activity and endogenous glycine betaine (GB) content in loquats treated with LTC were significantly higher than those in control fruit. Moreover, LTC treatment induced activities of energy metabolism-associated enzymes, including H(+)-adenosine triphosphatase, Ca(2+)-adenosine triphosphatase, succinic dehydrogenase, and cytochrome c oxidase. LTC treatment triggered obviously higher levels of adenosine triphosphate (ATP) content and energy charge in loquat fruit. These results showed that LTC possibly alleviated chilling injury and enhanced chilling tolerance of loquat fruit by enhancing endogenous GB content and energy status.

  17. Acute hyperammonemia and systemic inflammation is associated with increased extracellular brain adenosine in rats

    Bjerring, Peter Nissen; Dale, Nicholas; Larsen, Fin Stolze


    Acute liver failure (ALF) can lead to brain edema, cerebral hyperperfusion and intracranial hypertension. These complications are thought to be mediated by hyperammonemia and inflammation leading to altered brain metabolism. As increased levels of adenosine degradation products have been found...... in brain tissue of patients with ALF we investigated whether hyperammonemia could induce adenosine release in brain tissue. Since adenosine is a potent vasodilator and modulator of cerebral metabolism we furthermore studied the effect of adenosine receptor ligands on intracranial pressure (ICP......) and cerebral blood flow (CBF). We measured the adenosine concentration with biosensors in rat brain slices exposed to ammonia and in a rat model with hyperammonemia and systemic inflammation. Exposure to ammonia in concentrations from 0.15-10 mM led to increases in the cortical adenosine concentration up to 18...

  18. Molecular Shape Analysis-Guided Virtual Screening Platform for Adenosine Kinase Inhibitors

    Bhutoria, Savita; Das, Ballari; Ghoshal, Nanda


    We propose a new application of molecular shape descriptors in hierarchical selection during virtual screening (VS). Here, a structure-based pharmacophore and docking-guided VS protocol have been evolved to identify inhibitors against adenosine kinase (AK). The knowledge gained on the shape requirements has been extrapolated in classifying active and inactive molecules against this target. This classification enabled us to pick the appropriate ligand conformation in the binding site. We have suggested a set of hierarchical filters for VS, from a simple molecular shape analysis (MSA) descriptor-based recursive models to docking scores. This approach permits a systematic study to understand the importance of spatial requirements and limitations for inhibitors against AK. Finally, the guidelines on how to select compounds for AK to achieve success have been highlighted. The utility of this approach has been suggested by giving an example of database screening for plausible active compounds. PMID:27478367

  19. Expression of human adenosine deaminase in mice reconstituted with retrovirus-transduced hematopoietic stem cells

    Wilson, J.M.; Danos, O.; Grossman, M.; Raulet, D.H.; Mulligan, R.C. (Massachusetts Institute of Technology, Cambridge (USA))


    Recombinant retroviruses encoding human adenosine deaminase have been used to infect murine hematopoietic stem cells. In bone marrow transplant recipients reconstituted with the genetically modified cells, human ADA was detected in peripheral blood mononuclear cells of the recipients for at least 6 months after transplantation. In animals analyzed in detail 4 months after transplantation, human ADA and proviral sequences were detected in all hematopoietic lineages; in several cases, human ADA activity exceeded the endogenous activity. These studies demonstrate the feasibility of introducing a functional human ADA gene into hematopoietic stem cells and obtaining expression in multiple hematopoietic lineages long after transplantation. This approach should be helpful in designing effective gene therapies for severe combined immunodeficiency syndromes in humans.

  20. Regulation of TrkB receptor translocation to lipid rafts by adenosine A2A receptors and its functional implications for BDNF-induced regulation of synaptic plasticity

    Assaife-Lopes, Natália; Sousa, Vasco C.; Pereira, Daniela B.; Ribeiro, Joaquim A.; Sebastião, Ana M.


    Brain-derived neurotrophic factor (BDNF) signalling is critical for neuronal development and transmission. Recruitment of TrkB receptors to lipid rafts has been shown to be necessary for the activation of specific signalling pathways and modulation of neurotransmitter release by BDNF. Since TrkB receptors are known to be modulated by adenosine A2A receptor activation, we hypothesized that activation of A2A receptors could influence TrkB receptor localization among different membrane microdoma...

  1. Interaction between Intrathecal Gabapentin and Adenosine in the Formalin Test of Rats

    Yoon, Myung Ha; Choi, Jeong Il; Park, Heon Chang; Bae, Hong Beom


    Spinal gabapentin and adenosine have been known to display an antinociceptive effect. We evaluated the nature of the interaction between gabapentin and adenosine in formalin-induced nociception at the spinal level. Male Sprague-Dawley rats were prepared for intrathecal catheterization. Pain was evoked by injection of formalin solution (5%, 50 µL) into the hindpaw. After examination of the effects of gabapentin and adenosine, the resulting interaction was investigated with isobolographic and f...

  2. Adenosine A2A Receptors Modulate Acute Injury and Neuroinflammation in Brain Ischemia.

    Felicita Pedata; Anna Maria Pugliese; Elisabetta Coppi; Ilaria Dettori; Giovanna Maraula; Lucrezia Cellai; Alessia Melani


    The extracellular concentration of adenosine in the brain increases dramatically during ischemia. Adenosine A2A receptor is expressed in neurons and glial cells and in inflammatory cells (lymphocytes and granulocytes). Recently, adenosine A2A receptor emerged as a potential therapeutic attractive target in ischemia. Ischemia is a multifactorial pathology characterized by different events evolving in the time. After ischemia the early massive increase of extracellular glutamate is followed by ...

  3. Endogenous adenosine and hemorrhagic shock: effects of caffeine administration or caffeine withdrawal.

    Conlay, L A; Evoniuk, G; Wurtman, R.J.


    Plasma adenosine concentrations doubled when rats were subjected to 90 min of profound hemorrhagic shock. Administration of caffeine (20 mg per kg of body weight), an adenosine-receptor antagonist, attenuated the hemorrhage-induced decrease in blood pressure. In contrast, chronic caffeine consumption (0.1% in drinking water), followed by a brief period of caffeine withdrawal, amplified the hypotensive response to hemorrhage. These data suggest that endogenous adenosine participates in the hyp...

  4. Rapid parallel flow cytometry assays of active GTPases using effector beads.

    Buranda, Tione; BasuRay, Soumik; Swanson, Scarlett; Agola, Jacob; Bondu, Virginie; Wandinger-Ness, Angela


    We describe a rapid assay for measuring the cellular activity of small guanine triphosphatases (GTPases) in response to a specific stimulus. Effector-functionalized beads are used to quantify in parallel multiple GTP-bound GTPases in the same cell lysate by flow cytometry. In a biologically relevant example, five different Ras family GTPases are shown for the first time to be involved in a concerted signaling cascade downstream of receptor ligation by Sin Nombre hantavirus.

  5. Topical adenosine increases the proportion of thick hair in Caucasian men with androgenetic alopecia.

    Iwabuchi, Tokuro; Ideta, Ritsuro; Ehama, Ritsuko; Yamanishi, Haruyo; Iino, Masato; Nakazawa, Yosuke; Kobayashi, Takashi; Ohyama, Manabu; Kishimoto, Jiro


    Adenosine is an effective treatment for androgenetic alopecia (AGA) in Japanese men and women. Adenosine exerts its effects by significantly increasing the proportion of thick hair. In this study, we assessed the clinical outcome of adenosine treatment for 6 months in 38 Caucasian men. The change in proportion of thick hair (≥60 μm) compared with baseline in the adenosine group was significantly higher than that in the placebo group (P thick hair in Caucasian men with AGA as well as in Japanese men and women.

  6. Adenosine Modulates the Oocyte Developmental Competence by Exposing Stages and Synthetic Blocking during In Vitro Maturation.

    Cheon, Yong-Pil


    Purine metabolism is known factor for nuclear maturation of oocytes through both follicle cells and oocyte itself. However, it is largely unknown the roles of purine metabolism in the oocyte competence for fertilization and early development. In this study, the effects of adenosine in oocyte competence for development were examined using adenosine and its synthetic inhibitors. Adenosine treatment from GV intact stage for 18 hr (fGV) caused of decrease the fertilization rate but of increase the cleavage rate compared from the other stage treatment groups. Hadacidin did not effect on fertilization rate but increased cleavage rate without stage specificity. Adenosine did not block the effects of hadacidin with the exception of fGV group. By the inhibition of purine synthetic pathways the fertilization rate was decreased in the fGV and fGVB groups but increased in the fMII group. Exogenous adenosine caused of decrease fertilization rate in the fGVB group but increase in the fMII group. Cleavage rate was dramatically increased in the adenosine treatment with synthetic inhibitors. It means that the metabolism of purine has stage specific effects on fertilization and cleavage. Exogenous adenosine had only can improve oocyte developmental competence when it treated at GV intact stage. On the other hand, endogenous synthesis in all maturation stage caused of increase the cleavage rate without effects on fertilization. These data suggest that adenosine at GV stage as a paracrine fashion and inhibitions of endogenous adenosine in all stage improve oocyte developmental competence..

  7. Selective adenosine A2A receptor agonists and antagonists protect against spinal cord injury through peripheral and central effects

    Esposito Emanuela


    Full Text Available Abstract Background Permanent functional deficits following spinal cord injury (SCI arise both from mechanical injury and from secondary tissue reactions involving inflammation. Enhanced release of adenosine and glutamate soon after SCI represents a component in the sequelae that may be responsible for resulting functional deficits. The role of adenosine A2A receptor in central ischemia/trauma is still to be elucidated. In our previous studies we have demonstrated that the adenosine A2A receptor-selective agonist CGS21680, systemically administered after SCI, protects from tissue damage, locomotor dysfunction and different inflammatory readouts. In this work we studied the effect of the adenosine A2A receptor antagonist SCH58261, systemically administered after SCI, on the same parameters. We investigated the hypothesis that the main action mechanism of agonists and antagonists is at peripheral or central sites. Methods Spinal trauma was induced by extradural compression of SC exposed via a four-level T5-T8 laminectomy in mouse. Three drug-dosing protocols were utilized: a short-term systemic administration by intraperitoneal injection, a chronic administration via osmotic minipump, and direct injection into the spinal cord. Results SCH58261, systemically administered (0.01 mg/kg intraperitoneal. 1, 6 and 10 hours after SCI, reduced demyelination and levels of TNF-α, Fas-L, PAR, Bax expression and activation of JNK mitogen-activated protein kinase (MAPK 24 hours after SCI. Chronic SCH58261 administration, by mini-osmotic pump delivery for 10 days, improved the neurological deficit up to 10 days after SCI. Adenosine A2A receptors are physiologically expressed in the spinal cord by astrocytes, microglia and oligodendrocytes. Soon after SCI (24 hours, these receptors showed enhanced expression in neurons. Both the A2A agonist and antagonist, administered intraperitoneally, reduced expression of the A2A receptor, ruling out the possibility that the

  8. The A3 adenosine receptor attenuates the calcium rise triggered by NMDA receptors in retinal ganglion cells.

    Zhang, Mei; Hu, Huiling; Zhang, Xiulan; Lu, Wennan; Lim, Jason; Eysteinsson, Thor; Jacobson, Kenneth A; Laties, Alan M; Mitchell, Claire H


    The A(3) adenosine receptor is emerging as an important regulator of neuronal signaling, and in some situations receptor stimulation can limit excitability. As the NMDA receptor frequently contributes to neuronal excitability, this study examined whether A(3) receptor activation could alter the calcium rise accompanying NMDA receptor stimulation. Calcium levels were determined from fura-2 imaging of isolated rat retinal ganglion cells as these neurons possess both receptor types. Brief application of glutamate or NMDA led to repeatable and reversible elevations of intracellular calcium. The A(3) agonist Cl-IB-MECA reduced the response to both glutamate and NMDA. While adenosine mimicked the effect of Cl-IB-MECA, the A(3) receptor antagonist MRS 1191 impeded the block by adenosine, implicating a role for the A(3) receptor in response to the natural agonist. The A(1) receptor antagonist DPCPX provided additional inhibition, implying a contribution from both A(1) and A(3) adenosine receptors. The novel A(3) agonist MRS 3558 (1'S,2'R,3'S,4'R,5'S)-4-(2-chloro-6-(3-chlorobenzylamino)-9H-purin-9-yl)-2,3-dihydroxy-N-methylbicyclo [3.1.0] hexane-1-carboxamide and mixed A(1)/A(3) agonist MRS 3630 (1'S,2'R,3'S,4'R,5'S)-4-(2-chloro-6-(cyclopentylamino)-9H-purin-9-yl)-2,3-dihydroxy-N-methylbicyclo [3.1.0] hexane-1-carboxamide also inhibited the calcium rise induced by NMDA. Low levels of MRS 3558 were particularly effective, with an IC(50) of 400 pM. In all cases, A(3) receptor stimulation inhibited only 30-50% of the calcium rise. In summary, stimulation of the A(3) adenosine receptor by either endogenous or synthesized agonists can limit the calcium rise accompanying NMDA receptor activation. It remains to be determined if partial block of the calcium rise by A(3) agonists can modify downstream responses to NMDA receptor stimulation.

  9. 腺苷及其受体对中性粒细胞在炎症中的作用与机制研究进展%Function and mechanism of adenosine and adenosine receptor on neutrophil during inflammation

    刘阳珷玥; 杨亭(综述); 赵力; 戴双双(审校)


    腺苷,作为机体内ATP代谢过程中的重要产物,通过激活腺苷受体(adenosine receptors, AR)(A1R、A2AR、A2BR、A3 R)参与了机体的许多生理及病理过程的调节。腺苷受体在体内的各个组织器官具有广泛的分布,在免疫系统尤为丰富,如中性粒细胞、巨噬细胞、淋巴细胞、树突状细胞均有较高表达。炎症,是机体抵御外来刺激的一种防御性免疫机制,过度的炎症反应则会对机体造成损伤,中性粒细胞是其中重要的参与者之一,腺苷及其受体的调节密切参与了其黏附、迁移、细菌杀伤作用、炎症介质产生、凋亡等过程,从而对炎症起到调节作用。%Adenosine, an important metabolic product of ATP, regulates many physiological and pathological processes through activating different adenosine receptors (A1R, A2AR, A2BR, A3R).There is a wide distribution of adenosine receptors in vari-ous tissues and organs particularly in immune system such as neutrophil, which is closely involved in the process of inflammation.In-flammation is defensive immune mechanism body resists external stimulus.The excessive inflammatory reaction will result in injury of the body in which neutrophils are one of the most important participants.The regulation of adenosine and its receptors on neutrophils plays an important role in adhesion, migration, bacterial killing effects, inflammatory mediators release, apoptosis and other processes.

  10. Role of Adenosine Receptor(s) in the Control of Vascular Tone in the Mouse Pudendal Artery.

    Labazi, Hicham; Tilley, Stephen L; Ledent, Catherine; Mustafa, S Jamal


    Activation of adenosine receptors (ARs) has been implicated in the modulation of renal and cardiovascular systems, as well as erectile functions. Recent studies suggest that adenosine-mediated regulation of erectile function is mainly mediated through A2BAR activation. However, no studies have been conducted to determine the contribution of AR subtype in the regulation of the vascular tone of the pudendal artery (PA), the major artery supplying and controlling blood flow to the penis. Our aim was to characterize the contribution of AR subtypes and identify signaling mechanisms involved in adenosine-mediated vascular tone regulation in the PA. We used a DMT wire myograph for muscle tension measurements in isolated PAs from wild-type, A2AAR knockout, A2BAR knockout, and A2A/A2BAR double-knockout mice. Real-time reverse transcription-polymerase chain reaction was used to determine the expression of the AR subtypes. Data from our pharmacologic and genetic approaches suggest that AR activation-mediated vasodilation in the PA is mediated by both the A2AAR and A2BAR, whereas neither the A1AR nor A3AR play a role in vascular tone regulation of the PA. In addition, we showed that A2AAR- and A2BAR-mediated vasorelaxation requires activation of nitric oxide and potassium channels; however, only the A2AAR-mediated response requires protein kinase A activation. Our data are complemented by mRNA expression showing the expression of all AR subtypes with the exception of the A3AR. AR signaling in the PA may play an important role in mediating erection and represent a promising therapeutic option for the treatment of erectile dysfunction.

  11. Caffeine prevents antihyperalgesic effect of gabapentin in an animal model of CRPS-I: evidence for the involvement of spinal adenosine A1 receptor.

    Martins, Daniel F; Prado, Marcos R B; Daruge-Neto, Eduardo; Batisti, Ana P; Emer, Aline A; Mazzardo-Martins, Leidiane; Santos, Adair R S; Piovezan, Anna P


    This study was designed to determine whether 3 weeks of gabapentin treatment is effective in alleviating neuropathic pain-like behavior in animal models of complex regional pain syndrome type-I and partial sciatic nerve ligation (PSNL). We investigated the contribution of adenosine subtypes to the antihyperalgesic effect of gabapentin by examining the effect of caffeine, a non-selective adenosine A1 and A2 receptor antagonist or 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), a selective adenosine A1 subtype receptor antagonist on this effect. Neuropathic pain was produced by unilateral prolonged hind paw ischemia and reperfusion (I/R) or PSNL procedures which resulted in stimulus-evoked mechanical hyperalgesia. After procedures, animals received gabapentin (10, 30, or 100 mg/kg intraperitoneal, respectively), caffeine (10 mg/kg intraperitoneal or 150 nmol intrathecally) or DPCPX (3 µg intrathecally) alone or in combination. Mice were tested for tactile mechanical hyperalgesia at 1, 2, and 3 weeks following procedures. Gabapentin produced dose-related inhibition of mechanical hyperalgesia over a 3-week period, and this effect was blocked by concomitant caffeine or DPCPX administration 1 week after injuries. The results of this study demonstrated that the mechanism through which gabapentin produces its effect may involve the activation of adenosine A1 subtype receptor.

  12. Effect of phentolamine on the hyperemic response to adenosine in patients with microvascular disease.

    Aarnoudse, Wilbert; Geven, Maartje; Barbato, Emanuele; Botman, Kees-joost; De Bruyne, Bernard; Pijls, Nico H J


    For accurate measurement of the fractional flow reserve (FFR) of the myocardium, the presence of maximum hyperemia is of paramount importance. It has been suggested that the hyperemic effect of the conventionally used hyperemic stimulus, adenosine, could be submaximal in patients who have microvascular dysfunction and that adding alpha-blocking agents could augment the hyperemic response in these patients. We studied the effect of the nonselective alpha-blocking agent phentolamine, which was administered in addition to adenosine after achieving hyperemia, in patients who had microvascular disease and those who did not. Thirty patients who were referred for percutaneous coronary intervention were selected. Of these 30 patients, 15 had strong indications for microvascular disease and 15 did not. FFR was measured using intracoronary adenosine, intravenous adenosine, and intracoronary papaverine before and after intracoronary administration of the nonselective alpha blocker phentolamine. In patients who did not have microvascular disease, no differences in hyperemic response to adenosine were noted, whether or not alpha blockade was given before adenosine administration; FFR levels before and after phentolamine were 0.76 and 0.75, respectively, using intracoronary adenosine (p = 0.10) and 0.75 and 0.74, respectively, using intravenous adenosine (p = 0.20). In contrast, in patients who had microvascular disease, some increase in hyperemic response was observed after administration of phentolamine; FFR levels decreased from 0.74 to 0.70 using intracoronary adenosine (p = 0.003) and from 0.75 to 0.72 using intravenous adenosine (p = 0.04). Although statistically significant, the observed further decrease in microvascular resistance after addition of phentolamine was small and did not affect clinical decision making in any patient. In conclusion, when measuring FFR, routinely adding an alpha-blocking agent to adenosine does not affect clinical decision making.

  13. Adenosine conjugated lipidic nanoparticles for enhanced tumor targeting.

    Swami, Rajan; Singh, Indu; Jeengar, Manish Kumar; Naidu, V G M; Khan, Wahid; Sistla, Ramakrishna


    Delivering chemotherapeutics by nanoparticles into tumor is impeded majorly by two factors: nonspecific targeting and inefficient penetration. Targeted delivery of anti-cancer agents solely to tumor cells introduces a smart strategy because it enhances the therapeutic index compared with untargeted drugs. The present study was performed to investigate the efficiency of adenosine (ADN) to target solid lipid nanoparticles (SLN) to over expressing adenosine receptor cell lines such as human breast cancer and prostate cancer (MCF-7 and DU-145 cells), respectively. SLN were prepared by emulsification and solvent evaporation process using docetaxel (DTX) as drug and were characterized by various techniques like dynamic light scattering, differential scanning calorimeter and transmission electron microscopy. DTX loaded SLNs were surface modified with ADN, an adenosine receptors ligand using carbodiimide coupling. Conjugation was confirmed using infrared spectroscopy and quantified using phenol-sulfuric acid method. Conjugated SLN were shown to have sustained drug release as compared to unconjugated nanoparticles and drug suspension. Compared with free DTX and unconjugated SLN, ADN conjugated SLN showed significantly higher cytotoxicity of loaded DTX, as evidenced by in vitro cell experiments. The IC50 was 0.41 μg/ml for native DTX, 0.30 μg/ml for unconjugated SLN formulation, and 0.09 μg/ml for ADN conjugated SLN formulation in MCF-7 cell lines. Whereas, in DU-145, there was 2 fold change in IC50 of ADN-SLN as compared to DTX. IC50 was found to be 0.44 μg/ml for free DTX, 0.39 μg/ml for unconjugated SLN and 0.22 μg/ml for ADN-SLN. Annexin assay and cell cycle analysis assay further substantiated the cell cytotoxicity. Fluorescent cell uptake and competitive ligand-receptor binding assay corroborated the receptor mediated endocytosis pathway indicated role of adenosine receptors in internalization of conjugated particles. Pharmacokinetic studies of lipidic

  14.   Adenosine-diphosphate (ADP) reduces infarct size and improves porcine heart function after myocardial infarction

    Bune, Laurids Touborg; Larsen, Jens Kjærgaard Rolighed; Thaning, Pia;


    (UTP) are both released during myocardial ischemia, influencing hemodynamics. Both mediate the release of tissue plasminogen activator (t-PA), which can reduce infarct size (IS). The objective of this study was to investigate whether exogenous ADP and UTP administration during reperfusion could reduce......Acute myocardial infarction continues to be a major cause of morbidity and mortality. Timely reperfusion can substantially improve outcomes and the administration of cardioprotective substances during reperfusion is therefore highly attractive. Adenosine diphosphate (ADP) and uridine-5-triphoshate...... myocardial IS and whether this correlated to t-PA release or improvements in hemodynamic responses. Hemodynamic variables and t-PA were measured in 22 pigs before, during, and after 45 min of left anterior coronary artery occlusion. During reperfusion, the pigs were randomized to 240 min of intracoronary...

  15. Adenosine Stimulate Proliferation and Migration in Triple Negative Breast Cancer Cells

    Fernandez-Gallardo, Miriam; González-Ramírez, Ricardo; Sandoval, Alejandro; Monjaraz, Eduardo


    Emerging evidence suggests that the adenosine (Ado) receptors may play crucial roles in tumor progression. Here, we show that Ado increases proliferation and migration in a triple negative breast cancer model, the MDA-MB 231 cell line. The use of specific agonists and antagonists evidenced that these effects depend on the activation of the A2B receptor, which then triggers an intracellular response mediated by the adenylate cyclase/PKA/cAMP signaling pathway. Ado also increases the expression of NaV1.5 channels, a potential biomarker in breast cancer. Together, these data suggest important roles of the A2B receptors and NaV1.5 channels in the Ado-induced increase in proliferation and migration of the MDA-MB 231 cells. PMID:27911956

  16. Relevance of adenosine deaminase as a marker for tubercu-lous pleural effusion in developing countries

    Rishad A; Patil BS; Das KK


    Objective:Relevance of estimation of pleural adenosine deaminase (PADA)and serum adenosine deaminase (SADA)levels in pleural effusion especially in cases of lymphocytic predominant exudative tubercular effu-sions.Methods:Fifty patients (33 male and 17 female;age:44.12 ±11.51 years)with pleural effusions were selected to assay adenosine deaminase (ADA)activity in pleural fluid and serum in adjunct to pleural fluid analysis.Effusions were individually classified as transudates or exudates after careful evaluation of all the biochemical parameters of pleural fluid and serum of patients and on the basis of Light's criteria.Cutoff val-ue for PADA was taken as 60U /L and that for pleural /serum ADA ratio (P/S ADA)was 1.8.Results:Four-ty-three patients had exudative effusions among which 38 patients had tuberculous pleural effusions and 5 had nontubercular effusions.7 cases were transudates.Mean PADA levels in tubercular group (78.95 ±25.32 U /L)were found to be much higher P =0.000 0)than nontubercular (23.00 ±5.22 U /L)group.SADA levels in tubercular group (31.05 ±6.42 U /L)were significantly higher (P =0.000 0)as compared to nontubercu-lar group (15.58 ±8.35 U /L).PADA cutoff at 60 U /L yielded sensitivity and specificity of 81.5% and 100% respectively,whereas P/S ADA ratio at 1.8 gave sensitivity and specificity of 84.2% and 75% respec-tively.A positive correlation (r =0.507,P =0.001 1)between PADA and SADA was found in tubercular group but no such correlation (r =0.302,P =0.340 7)was observed in nontubercular group.Conclusion:The measurement of ADA in tubercular pleural effusions has not only relevance but also a high diagnostic utility when other clinical and laboratory tests are either negative or confusing.

  17. The role of adenosine in exercise hyperaemia of the gracilis muscle in anaesthetized cats.

    Poucher, S M; Nowell, C G; Collis, M G


    1. A number of metabolites have been proposed to control the vascular tone of skeletal muscle during exercise. The present study was designed to investigate the role of adenosine in this response by determining the effect of the adenosine receptor antagonist 8-phenyltheophylline. 2. The gracilis muscle of anaesthetized cats was exposed and made to contract by stimulating the obturator nerve (at 1 Hz, 5 V, 0.1 ms) for 20 min. Gracilis muscle blood flow and tension were measured during exercise and for 20 min following exercise. Initially this was performed in each animal during the infusion of a vehicle solution (50% polyethylene glycol 400, 50% 0.1 M-NaOH, 0.1 ml min-1 I.V.). Exercise was then repeated during infusion of either further vehicle (group I), 8-phenyltheophylline (group II) or 3-propylxanthine (group III), both at 2.7 x 10(7) mol min-1 kg-1. 3. In group 1 (n = 4) gracilis muscle blood flow during the first exercise period increased by 47.5 +/- 11.3 ml min-1 (110 g)-1 and gracilis muscle tension by 8.6 +/- 1.3 kg (100 g muscle mass)-1 at 20 min of exercise. These responses were not significantly different when repeated. 4. In group II (n = 5), blood flow increased by 46.9 +/- 9.9 ml min-1 (100 g)-1 and tension by 6.5 +/- 0.7 kg (100 g muscle mass)-1 during vehicle infusion. Infusion of 8-phenyltheophylline at a rate which abolished the vasodilatation response to 2-chloroadenosine, significantly reduced the muscle blood flow increase to 19.8 +/- 2.7 ml min-1 (100 g muscle mass)-1 (P less than 0.05) but the tension response was unaffected (increased by 7.0 +/- 0.8 kg (100 g muscle mass)-1). 8-Phenyltheophylline did not affect gracilis muscle blood flow or tension at rest. 5. Administration of 3-propylxanthine, which did not modify the vasodilatation response to 2-chloroadenosine, failed to alter the vascular responses to muscle contraction. 6. These results suggest that activation of adenosine receptors can contribute to up to 40% of the vasodilatation

  18. Treatment of paroxysmal supraventricular tachycardia with intravenous injection of adenosine triphosphate.

    Saito, D.; Ueeda, M; Abe, Y.; Tani, H; Nakatsu, T.; Yoshida, H.; Haraoka, S; Nagashima, H


    Intravenous adenosine triphosphate rapidly terminated all 11 episodes of paroxysmal supraventricular tachycardia in 10 patients. Eight patients reported side effects but these resolved within 20 seconds and did not require treatment. Adenosine triphosphate is a suitable agent for the rapid termination of paroxysmal supraventricular tachycardia.

  19. The ischemic preconditioning effect of adenosine in patients with ischemic heart disease

    Berglund Margareta


    Full Text Available Abstract Introduction In vivo and in vitro evidence suggests that adenosine and its agonists play key roles in the process of ischemic preconditioning. The effects of low-dose adenosine infusion on ischemic preconditioning have not been thoroughly studied in humans. Aims We hypothesised that a low-dose adenosine infusion could reduce the ischemic burden evoked by physical exercise and improve the regional left ventricular (LV systolic function. Materials and methods We studied nine severely symptomatic male patients with severe coronary artery disease. Myocardial ischemia was induced by exercise on two separate occasions and quantified by Tissue Doppler Echocardiography. Prior to the exercise test, intravenous low-dose adenosine or placebo was infused over ten minutes according to a randomized, double blind, cross-over protocol. The LV walls were defined as ischemic if a reduction, no increment, or an increment of Results PSV increased from baseline to maximal exercise in non-ischemic walls both during placebo (P = 0.0001 and low-dose adenosine infusion (P = 0.0009. However, in the ischemic walls, PSV increased only during low-dose adenosine infusion (P = 0.001, while no changes in PSV occurred during placebo infusion (P = NS. Conclusion Low-dose adenosine infusion reduced the ischemic burden and improved LV regional systolic function in the ischemic walls of patients with exercise-induced myocardial ischemia, confirming that adenosine is a potential preconditioning agent in humans.

  20. Genetically Controlled Upregulation of Adenosine A(1) Receptor Expression Enhances the Survival of Primary Cortical Neurons

    Serchov, Tsvetan; Atas, Hasan-Cem; Normann, Claus; van Calker, Dietrich; Biber, Knut


    Adenosine has a key endogenous neuroprotective role in the brain, predominantly mediated by the adenosine A(1) receptor (A(1)R). This has been mainly explored using pharmacological tools and/or receptor knockout mice strains. It has long been suggested that the neuroprotective effects of A(1)R are i

  1. Nafion-CNT coated carbon-fiber microelectrodes for enhanced detection of adenosine.

    Ross, Ashley E; Venton, B Jill


    Adenosine is a neuromodulator that regulates neurotransmission. Adenosine can be monitored using fast-scan cyclic voltammetry at carbon-fiber microelectrodes and ATP is a possible interferent in vivo because the electroactive moiety, adenine, is the same for both molecules. In this study, we investigated carbon-fiber microelectrodes coated with Nafion and carbon nanotubes (CNTs) to enhance the sensitivity of adenosine and decrease interference by ATP. Electrodes coated in 0.05 mg mL(-1) CNTs in Nafion had a 4.2 ± 0.2 fold increase in current for adenosine, twice as large as for Nafion alone. Nafion-CNT electrodes were 6 times more sensitive to adenosine than ATP. The Nafion-CNT coating did not slow the temporal response of the electrode. Comparing different purine bases shows that the presence of an amine group enhances sensitivity and that purines with carbonyl groups, such as guanine and hypoxanthine, do not have as great an enhancement after Nafion-CNT coating. The ribose group provides additional sensitivity enhancement for adenosine over adenine. The Nafion-CNT modified electrodes exhibited significantly more current for adenosine than ATP in brain slices. Therefore, Nafion-CNT modified electrodes are useful for sensitive, selective detection of adenosine in biological samples.

  2. The effect of circulating adenosine on cerebral haemodynamics and headache generation in healthy subjects

    Birk, S; Petersen, K.A.; Kruuse, Christina Rostrup


    been investigated in man and reports regarding the effect of intravenous adenosine on cerebral blood flow are conflicting. Twelve healthy participants received adenosine 80, 120 microg kg(-1) min(-1) and placebo intravenously for 20 min, in a double-blind, three-way, crossover, randomized design...

  3. Different Modulating Effects of Adenosine on Neonatal and Adult Polymorphonuclear Leukocytes

    Pei-Chen Hou


    Full Text Available Polymorphonuclear leukocytes (PMNs are the major leukocytes in the circulation and play an important role in host defense. Intact PMN functions include adhesion, migration, phagocytosis, and reactive oxygen species (ROS release. It has been known for a long time that adenosine can function as a modulator of adult PMN functions. Neonatal plasma has a higher adenosine level than that of adults; however, little is known about the modulating effects of adenosine on neonatal PMNs. The aim of this study was to investigate the effects of adenosine on neonatal PMN functions. We found that neonatal PMNs had impaired adhesion, chemotaxis, and ROS production abilities, but not phagocytosis compared to adult PMNs. As with adult PMNs, adenosine could suppress the CD11b expressions of neonatal PMNs, but had no significant suppressive effect on phagocytosis. In contrast to adult PMNs, adenosine did not significantly suppress chemotaxis and ROS production of neonatal PMNs. This may be due to impaired phagocyte reactions and a poor neonatal PMN response to adenosine. Adenosine may not be a good strategy for the treatment of neonatal sepsis because of impaired phagocyte reactions and poor response.

  4. Adenosine signaling and the energetic costs of induced immunity.

    Brian P Lazzaro


    Full Text Available Life history theory predicts that trait evolution should be constrained by competing physiological demands on an organism. Immune defense provides a classic example in which immune responses are presumed to be costly and therefore come at the expense of other traits related to fitness. One strategy for mitigating the costs of expensive traits is to render them inducible, such that the cost is paid only when the trait is utilized. In the current issue of PLOS Biology, Bajgar and colleagues elegantly demonstrate the energetic and life history cost of the immune response that Drosophila melanogaster larvae induce after infection by the parasitoid wasp Leptopilina boulardi. These authors show that infection-induced proliferation of defensive blood cells commands a diversion of dietary carbon away from somatic growth and development, with simple sugars instead being shunted to the hematopoetic organ for rapid conversion into the raw energy required for cell proliferation. This metabolic shift results in a 15% delay in the development of the infected larva and is mediated by adenosine signaling between the hematopoietic organ and the central metabolic control organ of the host fly. The adenosine signal thus allows D. melanogaster to rapidly marshal the energy needed for effective defense and to pay the cost of immunity only when infected.

  5. Adenosine Deaminase Deficiency - More Than Just an Immunodeficiency

    Kathryn Victoria Whitmore


    Full Text Available Adenosine deaminase (ADA deficiency is best known as a form of severe combined immunodeficiency (SCID which results from mutations in the gene encoding adenosine deaminase. Affected patients present with clinical and immunological manifestations typical of a severe combined immunodeficiency. Therapies are currently available that can that target these immunological disturbances and treated patients show varying degrees of clinical improvement. However, there is now a growing body of evidence that deficiency of ADA has significant impact on non-immunological organ systems. This review will outline the impact of ADA deficiency on various organ systems, starting with the well understood immunological abnormalities. We will discuss possible pathogenic mechanisms and also highlight ways in which current treatments could be improved. In doing so, we aim to present ADA deficiency as more than an immunodeficiency and suggest that it should be recognized as a systemic metabolic disorder that affects multiple organ systems. Only by fully understanding ADA deficiency and its manifestations in all organ systems can we aim to deliver therapies that will correct all the clinical consequences.

  6. Adenosine Amine Congener as a Cochlear Rescue Agent

    Srdjan M. Vlajkovic


    Full Text Available We have previously shown that adenosine amine congener (ADAC, a selective A1 adenosine receptor agonist, can ameliorate noise- and cisplatin-induced cochlear injury. Here we demonstrate the dose-dependent rescue effects of ADAC on noise-induced cochlear injury in a rat model and establish the time window for treatment. Methods. ADAC (25–300 μg/kg was administered intraperitoneally to Wistar rats (8–10 weeks old at intervals (6–72 hours after exposure to traumatic noise (8–16 kHz, 110 dB sound pressure level, 2 hours. Hearing sensitivity was assessed using auditory brainstem responses (ABR before and 12 days after noise exposure. Pharmacokinetic studies investigated ADAC concentrations in plasma after systemic (intravenous administration. Results. ADAC was most effective in the first 24 hours after noise exposure at doses >50 μg/kg, providing up to 21 dB protection (averaged across 8–28 kHz. Pharmacokinetic studies demonstrated a short (5 min half-life of ADAC in plasma after intravenous administration without detection of degradation products. Conclusion. Our data show that ADAC mitigates noise-induced hearing loss in a dose- and time-dependent manner, but further studies are required to establish its translation as a clinical otological treatment.

  7. The Effects of the Adenosine Receptor Antagonists on the Reverse of Cardiovascular Toxic Effects Induced by Citalopram In-Vivo Rat Model of Poisoning

    Büyükdeligöz, Müjgan; Hocaoğlu, Nil; Oransay, Kubilay; Tunçok, Yeşim; Kalkan, Şule


    Background: Citalopram is a selective serotonin reuptake inhibitor that requires routine cardiac monitoring to prevent a toxic dose. Prolongation of the QT interval has been observed in acute citalopram poisoning. Our previous experimental study showed that citalopram may be lead to QT prolongation by stimulating adenosine A1 receptors without affecting the release of adenosine. Aims: We examined the effects of adenosine receptor antagonists in reversing the cardiovascular toxic effects induced by citalopram in rats. Study Design: Animal experimentation. Methods: Rats were divided into three groups randomly (n=7 for each group). Sodium cromoglycate (20 mg/kg) was administered to all rats to inhibit adenosine A3 receptor mast cell activation. Citalopram toxicity was achieved by citalopram infusion (4 mg/kg/min) for 20 minutes. After citalopram infusion, in the control group (Group 1), rats were given an infusion of dextrose solution for 60 minutes. In treatment groups, the selective adenosine A1 antagonist DPCPX (Group 2, 8-cyclopentyl-1,3-dipropylxanthine, 20 μg/kg/min) or the selective A2a antagonist CSC (Group 3, 8-(3-chlorostyryl)caffeine, 24 μg/kg/min) was infused for 60 minutes. Mean arterial pressure (MAP), heart rate (HR), QRS duration and QT interval measurements were followed during the experiment period. Statistical analysis was performed by ANOVA followed by Tukey’s multiple comparison tests. Results: Citalopram infusion reduced MAP and HR and prolonged the QT interval. It did not cause any significant difference in QRS duration in any group. When compared to the control group, DPCPX after citalopram infusion shortened the prolongation of the QT interval after 40, 50 and 60 minutes (p<0.01). DPCPX infusion shortened the prolongation of the QT interval at 60 minutes compared with the CSC group (p<0.05). CSC infusion shortened the prolongation of the QT at 60 minutes compared with the control group (p<0.05). Conclusion: DPCPX improved QT interval

  8. Reconstruction of the adenosine system by bone marrow-derived mesenchymal stem cell transplantation

    Huicong Kang; Qi Hu; Xiaoyan Liu; Yinhe Liu; Feng Xu; Xiang Li; Suiqiang Zhu


    In the present study, we transplanted bone marrow-derived mesenchymal stem cells into the CA3 area of the hippocampus of chronic epilepsy rats kindled by lithium chloride-pilocarpine. Immunofluorescence and western blotting revealed an increase in adenosine A1 receptor expression and a decrease in adenosine A2a receptor expression in the brain tissues of epileptic rats 3 months after transplantation. Moreover, the imbalance in the A1 adenosine receptor/A2a adenosine receptor ratio was improved. Electroencephalograms showed that frequency and amplitude of spikes in the hippocampus and frontal lobe were reduced. These results suggested that mesenchymal stem cell transplantation can reconstruct the normal function of the adenosine system in the brain and greatly improve epileptiform discharges.

  9. Comparison of exogenous adenosine and voluntary exercise on human skeletal muscle perfusion and perfusion heterogeneity

    Heinonen, Ilkka H.A.; Kemppainen, Jukka; Kaskinoro, Kimmo;


    femoral artery infusion of adenosine (1 mg * min(-1) * litre thigh volume(-1)), which has previously been shown to induce maximal whole thigh blood flow of ~8 L/min. This response was compared to the blood flow induced by moderate-high intensity one-leg dynamic knee extension exercise. Adenosine increased...... muscle. Additionally, it remains to be determined what proportion of adenosine-induced flow elevation is specifically directed to muscle only. In the present study we measured thigh muscle capillary nutritive blood flow in nine healthy young men using positron emission tomography at rest and during...... muscle blood flow on average to 40 +/- 7 ml. min(-1) per 100g(-1) of muscle and an aggregate value of 2.3 +/- 0.6 L * min(-1) for the whole thigh musculature. Adenosine also induced a substantial change in blood flow distribution within individuals. Muscle blood flow during adenosine infusion...

  10. Interstitial and plasma adenosine stimulate nitric oxide and prostacyclin formation in human skeletal muscle

    Nyberg, Michael Permin; Mortensen, Stefan Peter; Thaning, Pia;


    One major unresolved issue in muscle blood flow regulation is that of the role of circulating versus interstitial vasodilatory compounds. The present study determined adenosine-induced formation of NO and prostacyclin in the human muscle interstitium versus in femoral venous plasma to elucidate....... In young healthy humans, microdialysate was collected at rest, during arterial infusion of adenosine, and during interstitial infusion of adenosine through microdialysis probes inserted into musculus vastus lateralis. Muscle interstitial NO and prostacyclin increased with arterial and interstitial infusion...... levels. These findings provide novel insight into the role of adenosine in skeletal muscle blood flow regulation and vascular function by revealing that both interstitial and plasma adenosine have a stimulatory effect on NO and prostacyclin formation. In addition, both skeletal muscle and microvascular...

  11. 2-(1-Hexyn-1-yl)adenosine-induced intraocular hypertension is mediated via K+ channel opening through adenosine A2A receptor in rabbits.

    Konno, Takashi; Uchibori, Takehiro; Nagai, Akihiko; Kogi, Kentaro; Nakahata, Norimichi


    The present study was performed to clarify the mechanism of change in intraocular pressure by 2-(1-hexyn-1-yl)adenosine (2-H-Ado), a selective adenosine A2 receptor agonist, in rabbits. 2-H-Ado (0.1%, 50 microl)-induced ocular hypertension (E(max): 7.7 mm Hg) was inhibited by an adenosine A2A receptor antagonist 1,3,7-trimethyl-8-(3-chlorostyryl)xanthine, ATP-sensitive K+ channel blocker glibenclamide or 5-hydroxydecanoic acid, but not by an adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine, an adenosine A2B receptor antagonist alloxazine or a cyclooxygenase inhibitor indomethacin. The outflow facility induced by 2-H-Ado seems to be independent of increase in intraocular pressure or ATP-sensitive K+ channel. In contrast, the recovery rate in intraocular pressure decreased by hypertonic saline was accelerated by 2-H-Ado, and this response was dependent on ATP-sensitive K+ channel. These results suggest that 2-H-Ado-induced ocular hypertension is mediated via K+ channel opening through adenosine A2A receptor, and this is probably due to aqueous formation, but independent of change in outflow facility or prostaglandin production.

  12. Autophagy occurs within an hour of adenosine triphosphate treatment after nerve cell damage:the neuroprotective effects of adenosine triphosphate against apoptosis

    Na Lu; Baoying Wang; Xiaohui Deng; Honggang Zhao; Yong Wang; Dongliang Li


    After hypoxia, ischemia, or inlfammatory injuries to the central nervous system, the damaged cells release a large amount of adenosine triphosphate, which may cause secondary neuronal death. Autophagy is a form of cell death that also has neuroprotective effects. Cell Counting Kit assay, monodansylcadaverine staining, lfow cytometry, western blotting, and real-time PCR were used to determine the effects of exogenous adenosine triphosphate treatment at different concentrations (2, 4, 6, 8, 10 mmol/L) over time (1, 2, 3, and 6 hours) on the apoptosis and autophagy of SH-SY5Y cells. High concentrations of extracellular adenosine triphosphate induced autophagy and apoptosis of SH-SY5Y cells. The enhanced autophagy ifrst appeared, and peaked at 1 hour after treatment with adenosine triphosphate. Cell apoptosis peaked at 3 hours, and persisted through 6 hours. With prolonged exposure to the adenosine triphosphate treatment, the fraction of apoptotic cells increased. These data suggest that the SH-SY5Y neural cells initiated autophagy against apoptosis within an hour of adenosine triphosphate treatment to protect themselves against injury.

  13. Sawhorse waveform voltammetry for selective detection of adenosine, ATP, and hydrogen peroxide.

    Ross, Ashley E; Venton, B Jill


    Fast-scan cyclic voltammetry (FSCV) is an electrochemistry technique which allows subsecond detection of neurotransmitters in vivo. Adenosine detection using FSCV has become increasingly popular but can be difficult because of interfering agents which oxidize at or near the same potential as adenosine. Triangle shaped waveforms are traditionally used for FSCV, but modified waveforms have been introduced to maximize analyte sensitivity and provide stability at high scan rates. Here, a modified sawhorse waveform was used to maximize the time for adenosine oxidation and to manipulate the shapes of cyclic voltammograms (CVs) of analytes which oxidize at the switching potential. The optimized waveform consists of scanning at 400 V/s from -0.4 to 1.35 V and holding briefly for 1.0 ms followed by a ramp back down to -0.4 V. This waveform allows the use of a lower switching potential for adenosine detection. Hydrogen peroxide and ATP also oxidize at the switching potential and can interfere with adenosine measurements in vivo; however, their CVs were altered with the sawhorse waveform and they could be distinguished from adenosine. Principal component analysis (PCA) was used to determine that the sawhorse waveform was better than the triangle waveform at discriminating between adenosine, hydrogen peroxide, and ATP. In slices, mechanically evoked adenosine was identified with PCA and changes in the ratio of ATP to adenosine were observed after manipulation of ATP metabolism by POM-1. The sawhorse waveform is useful for adenosine, hydrogen peroxide, and ATP discrimination and will facilitate more confident measurements of these analytes in vivo.

  14. 75 FR 8981 - Prospective Grant of Exclusive License: Treatment of Glaucoma by Administration of Adenosine A3...


    ... Glaucoma by Administration of Adenosine A3 Antagonists AGENCY: National Institutes of Health, Public Health.../092,292, entitled ``A3 Adenosine Receptor Antagonists,'' filed July 10, 1998 , PCT Application PCT/US99/ 15562, entitled''A3 Adenosine Receptor Antagonists,'' filed July 2, 1999 , U.S. Patent...

  15. Wireless fast-scan cyclic voltammetry to monitor adenosine in patients with essential tremor during deep brain stimulation.

    Chang, Su-Youne; Kim, Inyong; Marsh, Michael P; Jang, Dong Pyo; Hwang, Sun-Chul; Van Gompel, Jamie J; Goerss, Stephan J; Kimble, Christopher J; Bennet, Kevin E; Garris, Paul A; Blaha, Charles D; Lee, Kendall H


    Essential tremor is often markedly reduced during deep brain stimulation simply by implanting the stimulating electrode before activating neurostimulation. Referred to as the microthalamotomy effect, the mechanisms of this unexpected consequence are thought to be related to microlesioning targeted brain tissue, that is, a microscopic version of tissue ablation in thalamotomy. An alternate possibility is that implanting the electrode induces immediate neurochemical release. Herein, we report the experiment performing with real-time fast-scan cyclic voltammetry to quantify neurotransmitter concentrations in human subjects with essential tremor during deep brain stimulation. The results show that the microthalamotomy effect is accompanied by local neurochemical changes, including adenosine release.

  16. Cyclic adenosine 3',5'-monophosphate and germination of sporangiospores from the fungus Mucor.

    Orlowski, M


    Cyclic adenosine 3',5'-monophosphate (cAMP) metabolism was examined in germinating sporangiospores of Mucor genevensis and Mucor mucedo. Exogenous cAMP prevented normal hyphal development from sporangiospores. Internal pools of cAMP fluctuated profoundly during development. Spherical growth of the spores was characterized by large pools of cAMP whereas germ tube emergence and hyphal elongation were characterized by small pools of cAMP. These observations suggest a possible role for cAMP in sporangiospore germination. Adenylate cyclase activities fluctuated significantly during germination with maximum values attained during spherical growth. In contrast, cAMP phosphodiesterase activities remained constant throughout germination. Internal cAMP levels may therefore be regulated by adjustment of adenylate cyclase activities. The binding of cAMP by soluble cell proteins was measured. cAMP-binding activity changed greatly during germination. Dormant and spherically growing spores possessed the highest activities. Developing hyphae contained the lowest activities. Use of the photoaffinity label, 8-azido-[32P]cAMP, in conjunction with sodium dodecyl sulfate-polyacrylamide-gel electrophoresis allowed the identification of a small population of morphogenetic-stage-specific proteins which bind cAMP and may be of regulatory significance to development.

  17. Overexpression of adenosine A2A receptors in rats: effects on depression, locomotion and anxiety

    Joana E Coelho


    Full Text Available Adenosine A2A receptors (A2AR are a sub-type of receptors enriched in basal ganglia, activated by the neuromodulator adenosine, which interact with dopamine D2 receptors. Although this reciprocal antagonistic interaction is well established in motor function, the outcome in dopamine-related behaviors remains uncertain, in particular in depression and anxiety. We have demonstrated an upsurge of A2AR associated to aging and chronic stress. Furthermore, Alzheimer’s disease patients present A2AR accumulation in cortical areas together with depressive signs. We now tested the impact of overexpressing A2AR in forebrain neurons on dopamine related behavior, namely depression. Adult male rats overexpressing human A2AR under the control of CaMKII promoter [Tg(CaMKII-hA2AR] and aged-matched wild-types (WT of the same strain (Sprague-Dawley were studied. The forced swimming test (FST, sucrose preference test (SPT and the open-field test (OFT were performed to evaluate behavioral despair, anhedonia, locomotion and anxiety. Tg(CaMKII-hA2AR animals spent more time floating and less time swimming in the FST and presented a decreased sucrose preference at 48h in the SPT. They also covered higher distances in the OFT and spent more time in the central zone than the WT. The results indicate that Tg(CaMKII-hA2AR rats exhibit depressive-like behavior, hyperlocomotion and altered exploratory behavior. This A2AR overexpression may explain the depressive signs found in aging, chronic stress and Alzheimer’s disease.

  18. Dynamic changes of adenosine triphosphate enzyme activity in encephalon tissue of rat with posttraumatic stress disorder psycho and behaviour abnormity%创伤后应激障碍样情感行为异常大鼠脑组织ATP酶活性的动态变化



    AIM:To discuss the pathophysiology basis of posttraumatic stress disorder(PTSD like) psycho and behaviour abnormity in attempt to provide a new method in treatments. METHODS:Seventy two male Wistar rats were randomly divided into three groups:hippocampus under threshold electric stimulation group(SE,n=32),hippocampus electrode burying control group(CE,n=32) and normal control group(NC,n=8).Hippocampus were continuously stimulated by constant monopulse electricity,with 25 Hz frequency,1 ms wave length,10 s cluster length,7 min cluster interval and 100 μ A strength under eclampsia threshold. The enzymatic activity changes of Na+ K+ adenosine triphosphate enzyme(ATPase) and Ca2+ ATPase in hippocampal homogenate of the experimental animals and mitochondria were detected in quantitation.RESULTS:The enzymatic activity of Na+-K+-ATPase in hippocampus mitochondria decreased obviously(0.56±0.15)mmol/(kg·s)(F=4.348,P<0.01) in under-threshold electric stimulation group atfer 12 hours of electric timulations as well as(0.61±0.17) mmol/(kg·s) (P<0.05) after 48 hours,which were significantly lower than NC group (0.84±0.22) mmol/(kg·s) the enzymatic activity of Ca2+-ATPase in hippocampus mitochondria also decreased obviously into (0.53±0.14) mmol/(kg·s) (F=4.999,P<0.05) after 24 hours of electric stimulations as well as (0.60±0.16) mmol/(kg·s) after 72 hours, which were significantly lower than NC group (0.83±0.22) mmol/(kg·s).CONCLUSION:Functional damages of the hippocampus, especially the Na K pump and Ca2+ pump in hippocampal mitochondria may have an important significance in the occurrence and development of long term PTSD like psycho and behaviour abnormity in experimental animals.%目的:探讨创伤后应激障碍( posttraumatic stress disorder,PTSD)样精神与行为异常的病理生理基础,为其治疗途径提供新思路. 方法:将 72只雄性 Wistar大鼠随机分组为海马阈下电刺激组( SE, n=32)、海马电极埋植对照组( CE, n=32)

  19. Perinatal caffeine, acting on maternal adenosine A(1 receptors, causes long-lasting behavioral changes in mouse offspring.

    Olga Björklund

    Full Text Available BACKGROUND: There are lingering concerns about caffeine consumption during pregnancy or the early postnatal period, partly because there may be long-lasting behavioral changes after caffeine exposure early in life. METHODOLOGY/PRINCIPAL FINDINGS: We show that pregnant wild type (WT mice given modest doses of caffeine (0.3 g/l in drinking water gave birth to offspring that as adults exhibited increased locomotor activity in an open field. The offspring also responded to cocaine challenge with greater locomotor activity than mice not perinatally exposed to caffeine. We performed the same behavioral experiments on mice heterozygous for adenosine A(1 receptor gene (A(1RHz. In these mice signaling via adenosine A(1 receptors is reduced to about the same degree as after modest consumption of caffeine. A(1RHz mice had a behavioral profile similar to WT mice perinatally exposed to caffeine. Furthermore, it appeared that the mother's genotype, not offspring's, was critical for behavioral changes in adult offspring. Thus, if the mother partially lacked A(1 receptors the offspring displayed more hyperactivity and responded more strongly to cocaine stimulation as adults than did mice of a WT mother, regardless of their genotype. This indicates that long-term behavioral alterations in the offspring result from the maternal effect of caffeine, and not a direct effect on fetus. WT offspring from WT mother but having a A(1R Hz grandmother preserved higher locomotor response to cocaine. CONCLUSIONS/SIGNIFICANCE: We suggest that perinatal caffeine, by acting on adenosine A(1 receptors in the mother, causes long-lasting behavioral changes in the offspring that even manifest themselves in the second generation.

  20. A Study of Histology and Enzymatic Histochemistry on Rabbit's Retina in Acute Ocular Hypertension


    The changes of activities of enzymes relating to energy metabolism in rabbit's retina in acute ocular hypertension were observed. The activities of succinate dehydrogenase and adenosine triphosphatase were found to be reduced, while the activities of the lactatic dehydrognease and glucose-6-phosphatase increased. The results revealed the metabolic disturbance of energy in retina after acute ocular hypertension might be the underlying factors relating to the defects of the functions and structures of the...

  1. The genetics of feto-placental development: A study of acid phosphatase locus 1 and adenosine deaminase polymorphisms in a consecutive series of newborn infants

    Bergamaschi Antonio


    Full Text Available Abstract Background Acid phosphatase locus 1 and adenosine deaminase locus 1 polymorphisms show cooperative effects on glucose metabolism and immunological functions. The recent observation of cooperation between the two systems on susceptibility to repeated spontaneous miscarriage prompted us to search for possible interactional effects between these genes and the correlation between birth weight and placental weight. Deviation from a balanced development of the feto-placental unit has been found to be associated with perinatal morbidity and mortality and with cardiovascular diseases in adulthood. Methods We examined 400 consecutive newborns from the Caucasian population of Rome. Birth weight, placental weight, and gestational length were registered. Acid phosphatase locus 1 and adenosine deaminase locus 1 phenotypes were determined by starch gel electrophoresis and correlation analysis was performed by SPSS programs. Informed verbal consent to participate in the study was obtained from the mothers. Results Highly significant differences in birth weight-placental weight correlations were observed among acid phosphatase locus 1 phenotypes (p = 0.005. The correlation between birth weight and placental weight was markedly elevated in subjects carrying acid phosphatase locus 1 phenotypes with medium-low F isoform concentration (A, CA and CB phenotypes compared to those carrying acid phosphatase locus 1 phenotypes with medium-high F isoform concentration (BA and B phenotypes (p = 0.002. Environmental and developmental variables were found to exert a significant effect on birth weight-placental weight correlation in subjects with medium-high F isoform concentrations, but only a marginal effect was observed in those with medium-low F isoform concentrations. The correlation between birth weight and placental weight is higher among carriers of the adenosine deaminase locus 1 allele*2, which is associated with low activity, than in homozygous adenosine

  2. Observation on the activities of alpha-L-fucosidase,alkaline phosphatase and adenosine deaminase in pregnant women%妊娠女性α-L-岩藻糖苷酶、碱性磷酸酶和腺苷脱氨酶活性观察

    王玮玮; 孙伟才; 周惠玉


    目的:妊娠期女性观察血清α-L-岩藻糖苷酶(AFU)、碱性磷酸酶(ALP)及腺苷脱氨酶(ADA)活性的变化及其与妊娠周期的相关性。方法分别检测352例体检正常的不同妊娠期女性(妊娠组,其中早期妊娠210例、晚期妊娠142例)及322例因不孕就诊且尚未妊娠的体检正常的女性(非妊娠组)血清 AFU、ALP、ADA 活性。对所有对象均进行动态监测(早期妊娠组于妊娠第14、22、30及38周分别检测,晚期妊娠组于妊娠第38及40周检测,非妊娠组于就诊时、就诊后第8及24周检测)并做比较。结果妊娠组血清 ALP、AFU 活性明显高于非妊娠组(P =0.000),ADA 活性则低于非妊娠组(P =0.000)。晚期妊娠组血清 ALP、AFU 活性明显高于早期妊娠组(P =0.000),但 ADA 活性两组间差异无统计学意义(P >0.05)。对早期妊娠组的动态监测显示,随着妊娠周期的增加,AFU 及 ALP 活性逐渐升高,但 ADA 基本无变化。晚期妊娠组第38周及第40周血清 AFU、ALP、ADA 活性差异均无统计学意义(P >0.05)。对非妊娠组的动态监测显示,与就诊时比较,就诊后第8周 ALP、ADA 活性差异有统计学意义(P =0.000),就诊后第24周 ADA 活性差异有统计学意义;与就诊后第8周比较,就诊后第24周 ALP、AFU、ADA 活性差异均有统计学意义(P <0.05)。结论妊娠期女性血清 AFU 及 ALP 活性明显升高,且随孕周的增加,升高趋势越明显。%Objective To observe the activity changes of alpha-L-fucosidase (AFU),alkaline phosphatase (ALP) and adenosine deaminase (ADA)in pregnant women and their correlations with pregnant period.Methods The activities of AFU,ALP and ADA were determined in 352 pregnant women (210 cases with early-stage pregnancy and 142 cases with late-stage pregnancy)and 322 unpregnant women.Dynamic monitoring was performed (early-stage pregnancy

  3. Evidence for evoked release of adenosine and glutamate from cultured cerebellar granule cells

    Schousboe, A.; Frandsen, A.; Drejer, J. (Univ. of Copenhagen (Denmark))


    Evoked release of ({sup 3}H)-D-aspartate which labels the neurotransmitter glutamate pool in cultured cerebellar granule cells was compared with evoked release of adenosine from similar cultures. It was found that both adenosine and (3H)-D-aspartate could be released from the neurons in a calcium dependent manner after depolarization of the cells with either 10-100 microM glutamate or 50 mM KCl. Cultures of cerebellar granule cells treated with 50 microM kainate to eliminate GABAergic neurons behaved in the same way. This together with the observation that cultured astrocytes did not exhibit a calcium dependent, potassium stimulated adenosine release strongly suggest that cerebellar granule cells release adenosine in a neurotransmitter-like fashion together with glutamate which is the classical neurotransmitter of these neurons. Studies of the metabolism of adenosine showed that in the granule cells adenosine is rapidly metabolized to ATP, ADP, and AMP, but in spite of this, adenosine was found to be released preferential to ATP.

  4. Role of adenosine signalling and metabolism in β-cell regeneration

    Andersson, Olov, E-mail:


    Glucose homeostasis, which is controlled by the endocrine cells of the pancreas, is disrupted in both type I and type II diabetes. Deficiency in the number of insulin-producing β cells – a primary cause of type I diabetes and a secondary contributor of type II diabetes – leads to hyperglycemia and hence an increase in the need for insulin. Although diabetes can be controlled with insulin injections, a curative approach is needed. A potential approach to curing diabetes involves regenerating the β-cell mass, e.g. by increasing β-cell proliferation, survival, neogenesis or transdifferentiation. The nucleoside adenosine and its cognate nucleotide ATP have long been known to affect insulin secretion, but have more recently been shown to increase β-cell proliferation during homeostatic control and regeneration of the β-cell mass. Adenosine is also known to have anti-inflammatory properties, and agonism of adenosine receptors can promote the survival of β-cells in an inflammatory microenvironment. In this review, both intracellular and extracellular mechanisms of adenosine and ATP are discussed in terms of their established and putative effects on β-cell regeneration. - Highlights: • A potential way to cure diabetes is to regenerate the β-cell mass by promoting cell survival, proliferation or neogenesis. • Adenosine may promote β-cell regeneration through several cellular mechanisms. • Adenosine and its cognate nucleotide ATP can each promote β-cell proliferation. • Do adenosine and ATP interact in promoting β-cell proliferation?.

  5. The A2B adenosine receptor impairs the maturation and immunogenicity of dendritic cells.

    Wilson, Jeffrey M; Ross, William G; Agbai, Oma N; Frazier, Renea; Figler, Robert A; Rieger, Jayson; Linden, Joel; Ernst, Peter B


    The endogenous purine nucleoside adenosine is an important antiinflammatory mediator that contributes to the control of CD4(+) T cell responses. While adenosine clearly has direct effects on CD4(+) T cells, it remains to be determined whether actions on APC such as dendritic cells (DC) are also important. In this report we characterize DC maturation and function in BMDC stimulated with LPS in the presence or absence of the nonselective adenosine receptor agonist NECA (5'-N-ethylcarboxamidoadenosine). We found that NECA inhibited TNF-alpha and IL-12 in a concentration-dependent manner, whereas IL-10 production was increased. NECA-treated BMDC also expressed reduced levels of MHC class II and CD86 and were less effective at stimulating CD4(+) T cell proliferation and IL-2 production compared with BMDC exposed to vehicle control. Based on real-time RT-PCR, the A(2A) adenosine receptor (A(2A)AR) and A(2B)AR were the predominant adenosine receptors expressed in BMDC. Using adenosine receptor subtype selective antagonists and BMDC derived from A(2A)AR(-/-) and A(2B)AR(-/-)mice, it was shown that NECA modulates TNF-alpha, IL-12, IL-10, and CD86 responses predominantly via A(2B)AR. These data indicate that engagement of A(2B)AR modifies murine BMDC maturation and suggest that adenosine regulates CD4(+) T cell responses by selecting for DC with impaired immunogencity.

  6. Adenosine Monophosphate-Based Detection of Bacterial Spores

    Kern, Roger G.; Chen, Fei; Venkateswaran, Kasthuri; Hattori, Nori; Suzuki, Shigeya


    A method of rapid detection of bacterial spores is based on the discovery that a heat shock consisting of exposure to a temperature of 100 C for 10 minutes causes the complete release of adenosine monophosphate (AMP) from the spores. This method could be an alternative to the method described in the immediately preceding article. Unlike that method and related prior methods, the present method does not involve germination and cultivation; this feature is an important advantage because in cases in which the spores are those of pathogens, delays involved in germination and cultivation could increase risks of infection. Also, in comparison with other prior methods that do not involve germination, the present method affords greater sensitivity. At present, the method is embodied in a laboratory procedure, though it would be desirable to implement the method by means of a miniaturized apparatus in order to make it convenient and economical enough to encourage widespread use.

  7. Late-onset adenosine deaminase deficiency presenting with Heck's disease.

    Artac, Hasibe; Göktürk, Bahar; Bozdemir, Sefika Elmas; Toy, Hatice; van der Burg, Mirjam; Santisteban, Ines; Hershfield, Michael; Reisli, Ismail


    Focal epithelial hyperplasia, also known as Heck's disease, is a rare but distinctive entity of viral etiology with characteristic clinical and histopathological features. It is a benign, asymptomatic disease of the oral mucosa caused by human papilloma viruses (HPV). Previous studies postulated an association between these lesions and immunodeficiency. Genetic deficiency of adenosine deaminase (ADA) results in varying degrees of immunodeficiency, including neonatal onset severe combined immunodeficiency (ADA-SCID), and milder, later onset immunodeficiency. We report a 12-year-old girl with the late onset-ADA deficiency presenting with Heck's disease. Our case report should draw attention to the possibility of immunodeficiency in patients with HPV-induced focal epithelial hyperplasia.

  8. Correlation study of adenosine deaminase and its isoenzymes in type 2 diabetes mellitus

    Sapkota, Lokendra Bahadur; Thapa, Sangita; Subedi, Nuwadatta


    Objective Adenosine deaminase (ADA) plays an important role in cell-mediated immunity and modulation of insulin activity. Its clinical and diagnostic significance in Nepalese type 2 diabetes is not yet characterized. So, this study's objective was to determine the isoenzymatic activities of ADA (ADA1, ADA2, and total ADA) and show its correlation with demographic, anthropometric, and biochemical characteristics of type 2 Nepalese subjects with diabetes. Research design and methods This is a hospital-based cross-sectional study including 80 type 2 diabetes mellitus (DM) patients and same number of age-matched and sex-matched healthy controls. Data were collected using preformed set of questionnaires and biochemical data were obtained from the laboratory analysis of the patient's blood samples. Statistical analysis was performed with SPSS V.20. Results A significantly higher (pdiabetic cases compared with controls. Serum ADA activities were significantly higher in cases compared with controls (pdiabetes. Conclusions Serum ADA activities were significantly higher in type 2 diabetic patients compared with controls having significant positive correlation with glycemic parameters. Serum ADA and its isoenzymes could be used as biomarkers for assessing glycemic status in patients with type 2 DM.

  9. The adenosine A2B receptor is involved in anion secretion in human pancreatic duct Capan-1 epithelial cells

    Hayashi, M.; Inagaki, A.; Novak, Ivana;


    Adenosine modulates a wide variety of biological processes via adenosine receptors. In the exocrine pancreas, adenosine regulates transepithelial anion secretion in duct cells and is considered to play a role in acini-to-duct signaling. To identify the functional adenosine receptors and Cl− chann...

  10. Deficits in Endogenous Adenosine Formation by Ecto-5′-Nucleotidase/CD73 Impair Neuromuscular Transmission and Immune Competence in Experimental Autoimmune Myasthenia Gravis

    Laura Oliveira


    Full Text Available AMP dephosphorylation via ecto-5′-nucleotidase/CD73 is the rate limiting step to generate extracellular adenosine (ADO from released adenine nucleotides. ADO, via A2A receptors (A2ARs, is a potent modulator of neuromuscular and immunological responses. The pivotal role of ecto-5′-nucleotidase/CD73, in controlling extracellular ADO formation, prompted us to investigate its role in a rat model of experimental autoimmune myasthenia gravis (EAMG. Results show that CD4+CD25+FoxP3+ regulatory T cells express lower amounts of ecto-5′-nucleotidase/CD73 as compared to controls. Reduction of endogenous ADO formation might explain why proliferation of CD4+ T cells failed upon blocking A2A receptors activation with ZM241385 or adenosine deaminase in EAMG animals. Deficits in ADO also contribute to neuromuscular transmission failure in EAMG rats. Rehabilitation of A2AR-mediated immune suppression and facilitation of transmitter release were observed by incubating the cells with the nucleoside precursor, AMP. These findings, together with the characteristic increase in serum adenosine deaminase activity of MG patients, strengthen our hypothesis that the adenosinergic pathway may be dysfunctional in EAMG. Given that endogenous ADO formation is balanced by ecto-5′-nucleotidase/CD73 activity and that A2ARs exert a dual role to restore use-dependent neurocompetence and immune suppression in myasthenics, we hypothesize that stimulation of the two mechanisms may have therapeutic potential in MG.

  11. Adenine arabinoside inhibition of adenovirus replication enhanced by an adenosine deaminase inhibitor.

    Wigand, R


    The inhibition of adenovirus multiplication by adenine arabinoside was determined by yield reduction in one-step multiplication cycle. Inhibition was greatly enhanced by an adenosine deaminase inhibitor (2-deoxycoformycin) in concentrations down to 10 ng/ml. Adenovirus types from four subgroups showed similar results. However, the enhancing effect of adenosine deaminase inhibitor was great in HeLa cells, moderate in human fibroblasts, and negligible in Vero cells. This difference could be explained by different concentrations of adenosine deaminase found in cell homogenates.

  12. Adenosine A2A receptor binding profile of two antagonists, ST1535 and KW6002: consideration on the presence of atypical adenosine A2A binding sites

    Teresa Riccioni


    Full Text Available Adenosine A2A receptors seem to exist in typical (more in striatum and atypical (more in hippocampus and cortex subtypes. In the present study, we investigated the affinity of two adenosine A2A receptor antagonists, ST1535 [2 butyl -9-methyl-8-(2H-1,2,3-triazol 2-yl-9H-purin-6-xylamine] and KW6002 [(E-1,3-diethyl-8-(3,4-dimethoxystyryl-7-methyl-3,7-dihydro-1H-purine-2,6,dione] to the “typical” and “atypical” A2A binding sites. Affinity was determined by radioligand competition experiments in membranes from rat striatum and hippocampus. Displacement of the adenosine analog [3H]CGS21680 [2-p-(2-carboxyethylphenethyl-amino-5’-N-ethylcarbox-amidoadenosine] was evaluated in the absence or in the presence of either CSC [8-(3-chlorostyryl-caffeine], an adenosine A2A antagonist that pharmacologically isolates atypical binding sites, or DPCPX (8-cyclopentyl-1,3-dipropylxanthine, an adenosine A1 receptor antagonist that pharmacologically isolates typical binding site. ZM241385 [84-(2-[7-amino-2-(2-furyl [1,2,4]-triazol[2,3-a][1,3,5]triazin-5-yl amino]ethyl phenol] and SCH58261 [(5-amino-7-(β-phenylethyl-2-(8-furylpyrazolo(4,3-e-1,2,4-triazolo(1,5-c pyrimidine], two other adenosine A2A receptor antagonists, which were reported to differently bind to atypical and typical A2A receptors, were used as reference compounds. ST1535, KW6002, ZM241385 and SCH58261 displaced [3H]CGS21680 with higher affinity in striatum than in hippocampus. In hippocampus, no typical adenosine A2A binding was detected, and ST1535 was the only compound that occupied atypical A2A adenosine receptors. Present data are explained in terms of heteromeric association among adenosine A2A, A2B and A1 receptors, rather than with the presence of atypical A2A receptor subtype.

  13. The role of microorganisms in the degradation of adenosine triphosphate (ATP) in chill-stored common carp (Cyprinus carpio) fillets.

    Li, Dapeng; Zhang, Longteng; Song, Sijia; Wang, Zhiying; Kong, Chunli; Luo, Yongkang


    Biochemical and microbial changes after harvest strongly affect the final quality and shelf life of fish and fish products. In this study, the role of microbes in the degradation of adenosine triphosphate (ATP), and the origin of adenosine monophosphate deaminase (AMPD) and acid phosphatase (ACP) in common carp fillets during different stages of chilled storage (at 4°C) were investigated. The content of ATP, ADP, AMP, IMP, HxR, and Hx, the activity of AMPD and ACP, and the total count of viable, Aeromonas, Pseudomonas, H2S-producing bacteria, and lactic acid bacteria were examined. Results indicated that the population of microbial communities in control samples increased with storage time, and Pseudomonas peaked on the 10th day of storage. Changes in AMPD activity were less related to the abundance of microbes during the entire storage period. However, ACP was derived from both fish muscle and microbial secretion during the middle and late stages of storage. Degradation of ATP to IMP was not affected by spoilage bacteria, but the hydrolysis of IMP, and the transformation of HxR to Hx was affected considerably by the spoilage bacteria.

  14. Triazoloquinazolines as Human A3 Adenosine Receptor Antagonists: A QSAR Study

    Dae-Sil Lee


    Full Text Available Multiple linear regression analysis was performed on the quantitative structure-activity relationships (QSAR of the triazoloquinazoline adenosine antagonists for human A3receptors. The data set used for the QSAR analysis encompassed the activities of 33triazoloquinazoline derivatives and 72 physicochemical descriptors. A template moleculewas derived using the known molecular structure for one of the compounds when bound tothe human A2B receptor, in which the amide bond was in a cis-conformation. All the testcompounds were aligned to the template molecule. In order to identify a reasonable QSARequation to describe the data set, we developed a multiple linear regression program thatexamined every possible combination of descriptors. The QSAR equation derived from thisanalysis indicates that the spatial and electronic effects is greater than that of hydrophobiceffects in binding of the antagonists to the human A3 receptor. It also predicts that a largesterimol length parameter is advantageous to activity, whereas large sterimol widthparameters and fractional positive partial surface areas are nonadvatageous.

  15. Selective and potent adenosine A3 receptor antagonists by methoxyaryl substitution on the N-(2,6-diarylpyrimidin-4-yl)acetamide scaffold.

    Yaziji, Vicente; Rodríguez, David; Coelho, Alberto; García-Mera, Xerardo; El Maatougui, Abdelaziz; Brea, José; Loza, María Isabel; Cadavid, María Isabel; Gutiérrez-de-Terán, Hugo; Sotelo, Eddy


    The influence of diverse methoxyphenyl substitution patterns on the N-(2,6-diarylpyrimidin-4-yl)acetamide scaffold is herein explored in order to modulate the A(3) adenosine receptor antagonistic profile. As a result, novel ligands exhibiting excellent potency (K(i) on A(3) AR < 20 nM) and selectivity profiles (above 100-fold within the adenosine receptors family) are reported. Moreover, our joint theoretical and experimental approach allows the identification of novel pharmacophoric elements conferring A(3)AR selectivity, first established by a robust computational model and thereafter characterizing the most salient features of the structure-activity and structure-selectivity relationships in this series.

  16. Differential effects of selective adenosine antagonists on the effort-related impairments induced by dopamine D1 and D2 antagonism.

    Nunes, E J; Randall, P A; Santerre, J L; Given, A B; Sager, T N; Correa, M; Salamone, J D


    Mesolimbic dopamine (DA) is a critical component of the brain circuitry regulating behavioral activation and effort-related processes. Rats with impaired DA transmission reallocate their instrumental behavior away from food-reinforced tasks with high response requirements, and instead select less effortful food-seeking behaviors. Previous work showed that adenosine A(2A) antagonists can reverse the effects of DA D(2) antagonists on effort-related choice. However, less is known about the effects of adenosine A(1) antagonists. Despite anatomical data showing that A(1) and D(1) receptors are co-localized on the same striatal neurons, it is uncertain if A(1) antagonists can reverse the effects DA D(1) antagonists. The present work systematically compared the ability of adenosine A(1) and A(2A) receptor antagonists to reverse the effects of DA D(1) and D(2) antagonists on a concurrent lever pressing/feeding choice task. With this procedure, rats can choose between responding on a fixed ratio 5 lever-pressing schedule for a highly preferred food (i.e. high carbohydrate pellets) vs. approaching and consuming a less preferred rodent chow. The D(1) antagonist ecopipam (0.2 mg/kg i.p.) and the D(2) antagonist eticlopride (0.08 mg/kg i.p.) altered choice behavior, reducing lever pressing and increasing lab chow intake. Co-administration of the adenosine A(1) receptor antagonists 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 0.375, 0.75, and 1.5 mg/kg i.p.), and 8-cyclopentyltheophylline (CPT; 3.0, 6.0, 12.0 mg/kg i.p.) failed to reverse the effects of either the D(1) or D(2) antagonist. In contrast, the adenosine A(2A) antagonist KW-6002 (0.125, 0.25 and 0.5 mg/kg i.p.) was able to produce a robust reversal of the effects of eticlopride, as well as a mild partial reversal of the effects of ecopipam. Adenosine A(2A) and DA D(2) receptors interact to regulate effort-related choice behavior, which may have implications for the treatment of psychiatric symptoms such as psychomotor

  17. Balance between oxidative damage and proliferative potential in an experimental rat model of CCl4-induced cirrhosis: protective role of adenosine administration.

    Hernández-Muñoz, R; Díaz-Muñoz, M; López, V; López-Barrera, F; Yáñez, L; Vidrio, S; Aranda-Fraustro, A; Chagoya de Sánchez, V


    Oxidative stress and its consequent lipid peroxidation (LP) exert harmful effects, which have been currently involved in the generation of carbon tetrachloride-induced cirrhosis. However, the recent report that "physiological" LP can be associated with liver regeneration (LR) makes it necessary to discriminate between oxidative stress-induced and LR-associated LP. In rats rendered cirrhotic by continuous CCl4 administration for 4 weeks, moderate cell necrosis and fine fatty infiltration were found. The histological abnormalities were accompanied by increased LP, mainly accounted for by the microsomal and cytosolic fractions and evidence of oxidative stress (decreased hepatic glutathione content and changes in xanthine oxidase and pentose phosphate pathway activities). After 8 weeks, a micronodular cirrhosis developed, but oxidative stress was greatly attenuated, only persisting in the enhanced LP confined to microsomes. Simultaneous administration of adenosine, a reliable hepatoprotector that readily prevents the onset of liver fibrosis, was able to block the oxidative stress induced by the long-term CCl4 treatment but elicited a selective subcellular distribution of increased LP, similar to that found during LR. The adenosine-induced changes in liver LP (mainly in the nuclear fraction) correlated with an increased activity of thymidine kinase. Therefore, data suggest that adenosine-mediated preservation of energy availability and mitochondrial function could participate in preventing the onset of oxidative stress in cirrhotic rats. The latter could induce a successful liver recovery, curtailing the sequence of events leading to fibrogenesis.

  18. Evidence that the positive inotropic effects of the alkylxanthines are not due to adenosine receptor blockade.

    Collis, M. G.; Keddie, J. R.; Torr, S. R.


    We investigated the possibility that the positive inotropic effects of the alkylxanthines are due to adenosine receptor blockade. The potency of 8-phenyltheophylline, theophylline and enprofylline as adenosine antagonists was assessed in vitro, using the guinea-pig isolated atrium, and in vivo, using the anaesthetized dog. The order of potency of the alkylxanthines as antagonists of the negative inotropic response to 2-chloroadenosine in vitro, and of the hypotensive response to adenosine in vivo was 8-phenyltheophylline greater than theophylline greater than enprofylline. The order of potency of the alkylxanthines as positive inotropic and chronotropic agents in the anaesthetized dog was enprofylline greater than theophylline greater than 8-phenyltheophylline. The results of this study indicate that the inotropic effects of the alkylxanthines in the anaesthetized dog are not due to adenosine receptor blockade. PMID:6322898




    Bronchial hyperresponsiveness to adenosine 5'-monophosphate (AMP) was studied after allergen challenge in allergic asthmatic patients. Measurements were made with and without nedocromil sodium pretreatment. Nedocromil sodium inhibited both the early and late asthmatic reactions (P <.01). After aller

  20. Role of adenosine in regulating the heterogeneity of skeletal muscle blood flow during exercise in humans

    Heinonen, Ilkka; Nesterov, Sergey V; Kemppainen, Jukka;


    ) muscles during exercise, measured using positron emission tomography. In six healthy young women, BF was measured at rest and then during three incremental low and moderate intermittent isometric one-legged knee-extension exercise intensities without and with theophylline-induced nonselective adenosine...... exercise intensity in the QF muscle group. Adenosine seems to play a role in muscle BF heterogeneity even in the absence of changes in bulk BF at low and moderate one-leg intermittent isometric exercise intensities.......Evidence from both animal and human studies suggests that adenosine plays a role in the regulation of exercise hyperemia in skeletal muscle. We tested whether adenosine also plays a role in the regulation of blood flow (BF) distribution and heterogeneity among and within quadriceps femoris (QF...

  1. Cannabidiol, a non-psychotropic plant-derived cannabinoid, decreases inflammation in a murine model of acute lung injury: role for the adenosine A(2A) receptor.

    Ribeiro, Alison; Ferraz-de-Paula, Viviane; Pinheiro, Milena L; Vitoretti, Luana B; Mariano-Souza, Domenica P; Quinteiro-Filho, Wanderley M; Akamine, Adriana T; Almeida, Vinícius I; Quevedo, João; Dal-Pizzol, Felipe; Hallak, Jaime E; Zuardi, Antônio W; Crippa, José A; Palermo-Neto, João


    Acute lung injury is an inflammatory condition for which treatment is mainly supportive because effective therapies have not been developed. Cannabidiol, a non-psychotropic cannabinoid component of marijuana (Cannabis sativa), has potent immunosuppressive and anti-inflammatory properties. Therefore, we investigated the possible anti-inflammatory effect of cannabidiol in a murine model of acute lung injury. Analysis of total inflammatory cells and differential in bronchoalveolar lavage fluid was used to characterize leukocyte migration into the lungs; myeloperoxidase activity of lung tissue and albumin concentration in the bronchoalveolar lavage fluid were analyzed by colorimetric assays; cytokine/chemokine production in the bronchoalveolar lavage fluid was also analyzed by Cytometric Bead Arrays and Enzyme-Linked Immunosorbent Assay (ELISA). A single dose of cannabidiol (20mg/kg) administered prior to the induction of LPS (lipopolysaccharide)-induced acute lung injury decreases leukocyte (specifically neutrophil) migration into the lungs, albumin concentration in the bronchoalveolar lavage fluid, myeloperoxidase activity in the lung tissue, and production of pro-inflammatory cytokines (TNF and IL-6) and chemokines (MCP-1 and MIP-2) 1, 2, and 4days after the induction of LPS-induced acute lung injury. Additionally, adenosine A(2A) receptor is involved in the anti-inflammatory effects of cannabidiol on LPS-induced acute lung injury because ZM241385 (4-(2-[7-Amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol) (a highly selective antagonist of adenosine A(2A) receptor) abrogated all of the anti-inflammatory effects of cannabidiol previously described. Thus, we show that cannabidiol has anti-inflammatory effects in a murine model of acute lung injury and that this effect is most likely associated with an increase in the extracellular adenosine offer and signaling through adenosine A(2A) receptor.

  2. Role of adenosine A1 and A2A receptors in the alcohol withdrawal syndrome.

    Kaplan, G B; Bharmal, N H; Leite-Morris, K A; Adams, W R


    The role of adenosine receptor-mediated signaling was examined in the alcohol withdrawal syndrome. CD-1 mice received a liquid diet containing ethanol (6.7%, v/v) or a control liquid diet that were abruptly discontinued after 14 days of treatment. Mice consuming ethanol showed a progressive increase in signs of intoxication throughout the drinking period. Following abrupt discontinuation of ethanol diet, mice demonstrated reversible signs of handling-induced hyperexcitability that were maximal between 5-8 h. Withdrawing mice received treatment with adenosine receptor agonists at the onset of peak withdrawal (5.5 h) and withdrawal signs were blindly rated (during withdrawal hours 6 and 7). Adenosine A1-receptor agonist R-N6(phenylisopropyl)adenosine (0.15 and 0.3 mg/ kg) reduced withdrawal signs 0.5 and 1.5 h after drug administration in a dose-dependent fashion. Adenosine A2A-selective agonist 2-p-(2-carboxyethyl)phenylethyl-amino-5'-N-ethylcarboxamidoadenosine (0.3 mg/kg) reduced withdrawal signs at both time points. In ethanol-withdrawing mice, there were significant decreases in adenosine transporter sites in striatum without changes in cortex or cerebellum. In ethanol-withdrawing mice, there were no changes in adenosine A1 and A2A receptor concentrations in cortex, striatum, or cerebellum. There appears to be a role for adenosine A1 and A2A receptors in the treatment of the ethanol withdrawal syndrome. Published by Elsevier Science Inc.

  3. Sitagliptin attenuates sympathetic innervation via modulating reactive oxygen species and interstitial adenosine in infarcted rat hearts.

    Lee, Tsung-Ming; Chen, Wei-Ting; Yang, Chen-Chia; Lin, Shinn-Zong; Chang, Nen-Chung


    We investigated whether sitagliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor, attenuates arrhythmias through inhibiting nerve growth factor (NGF) expression in post-infarcted normoglycemic rats, focusing on adenosine and reactive oxygen species production. DPP-4 bound adenosine deaminase has been shown to catalyse extracellular adenosine to inosine. DPP-4 inhibitors increased adenosine levels by inhibiting the complex formation. Normoglycemic male Wistar rats were subjected to coronary ligation and then randomized to either saline or sitagliptin in in vivo and ex vivo studies. Post-infarction was associated with increased oxidative stress, as measured by myocardial superoxide, nitrotyrosine and dihydroethidium fluorescent staining. Measurement of myocardial norepinephrine levels revealed a significant elevation in vehicle-treated infarcted rats compared with sham. Compared with vehicle, infarcted rats treated with sitagliptin significantly increased interstitial adenosine levels and attenuated oxidative stress. Sympathetic hyperinnervation was blunted after administering sitagliptin, as assessed by immunofluorescent analysis and western blotting and real-time quantitative RT-PCR of NGF. Arrhythmic scores in the sitagliptin-treated infarcted rats were significantly lower than those in vehicle. Ex vivo studies showed a similar effect of erythro-9-(2-hydroxy-3-nonyl) adenine (an adenosine deaminase inhibitor) to sitagliptin on attenuated levels of superoxide and NGF. Furthermore, the beneficial effects of sitagliptin on superoxide anion production and NGF levels can be reversed by 8-cyclopentyl-1,3-dipropulxanthine (adenosine A1 receptor antagonist) and exogenous hypoxanthine. Sitagliptin protects ventricular arrhythmias by attenuating sympathetic innervation via adenosine A1 receptor and xanthine oxidase-dependent pathways, which converge through the attenuated formation of superoxide in the non-diabetic infarcted rats.

  4. Adenosine actions on CA1 pyramidal neurones in rat hippocampal slices.

    Greene, R W; Haas, H L


    Intracellular recordings with a bridge amplifier of CA1 pyramidal neurones in vitro were employed to study the mechanisms of action of exogenously applied adenosine in the hippocampal slice preparation of the rat. Adenosine enhanced the calcium-dependent, long-duration after-hyperpolarization (a.h.p.) at least in part by a reduction in the rate of decay of the a.h.p. Both the reduced rate of decay and that of the control can be described with a single exponential. Antagonism of the calcium-dependent potassium current (and as a result, the a.h.p.) by bath application of CdCl2 or intracellular injection of EGTA (ethyleneglycolbis-(beta-aminoethyl ether)N,N'-tetraacetic acid) did not reduce the adenosine-evoked hyperpolarization or decrease in input resistance. Similarly, TEA (tetraethylammonium), which antagonizes both the voltage- and calcium-sensitive, delayed, outward rectification, had no effect on the adenosine-evoked changes in resting membrane properties. Adenosine did not affect the early, transient, outward rectification. During exposure to 4-aminopyridine (4-AP) in concentrations sufficient to antagonize this early rectification, the changes in resting membrane properties evoked by adenosine were unaffected. We conclude that the enhancement of the a.h.p. and accommodation by adenosine may be mediated by a change in the regulation of intracellular calcium. However, the mechanism responsible for the hyperpolarization and decrease in input resistance evoked by adenosine is both calcium and voltage insensitive. Thus, it appears distinct from that mediating the enhancement of the a.h.p. and accommodation.

  5. The stimulatory adenosine receptor ADORA2B regulates serotonin (5-HT synthesis and release in oxygen-depleted EC cells in inflammatory bowel disease.

    Rikard Dammen

    Full Text Available OBJECTIVE: We recently demonstrated that hypoxia, a key feature of IBD, increases enterochromaffin (EC cell 5-HT secretion, which is also physiologically regulated by the ADORA2B mechanoreceptor. Since hypoxia is associated with increased extracellular adenosine, we wanted to examine whether this nucleotide amplifies HIF-1α-mediated 5-HT secretion. DESIGN: The effects of hypoxia were studied on IBD mucosa, isolated IBD-EC cells, isolated normal EC cells and the EC cell tumor derived cell line KRJ-1. Hypoxia (0.5% O2 was compared to NECA (adenosine agonist, MRS1754 (ADORA2B receptor antagonist and SCH442146 (ADORA2A antagonist on HIF signaling and 5-HT secretion. Antisense approaches were used to mechanistically evaluate EC cells in vitro. PCR and western blot were used to analyze transcript and protein levels of HIF-1α signaling and neuroendocrine cell function. An animal model of colitis was evaluated to confirm hypoxia:adenosine signaling in vivo. RESULTS: HIF-1α is upregulated in IBD mucosa and IBD-EC cells, the majority (~90% of which express an activated phenotype in situ. Hypoxia stimulated 5-HT release maximally at 30 mins, an effect amplified by NECA and selectively inhibited by MRS1754, through phosphorylation of TPH-1 and activation of VMAT-1. Transient transfection with Renilla luciferase under hypoxia transcriptional response element (HRE control identified that ADORA2B activated HIF-1α signaling under hypoxic conditions. Additional signaling pathways associated with hypoxia:adenosine included MAP kinase and CREB. Antisense approaches mechanistically confirmed that ADORA2B signaling was linked to these pathways and 5-HT release under hypoxic conditions. Hypoxia:adenosine activation which could be reversed by 5'-ASA treatment was confirmed in a TNBS-model. CONCLUSION: Hypoxia induced 5-HT synthesis and secretion is amplified by ADORA2B signaling via MAPK/CREB and TPH-1 activation. Targeting ADORA2s may decrease EC cell 5-HT

  6. Serum adenosine deaminase as oxidative stress marker in type 2 diabetes mellitus

    Shashikala Magadi Dasegowda


    Results: The study observed an increased level of serum adenosine deaminase, malondialdehyde and decreased levels of total antioxidant capacity in type 2 diabetes mellitus compared to controls. Serum adenosine deaminase levels in type 2 diabetics were 50.77 +/- 6.95 and in controls was 17.86 +/- 4.04. Serum Malondialdehyde levels in type 2 diabetics was 512.13 +/- 70.15 and in controls was 239.32 +/- 23.97. Serum total antioxidant levels in type 2 diabetics was 0.39+/-0.15 and in controls was 1.66+/-0.25. Positive correlation was seen between serum adenosine deaminase and malondialdehyde and it was statistically significant. Statistically significant negative correlation was seen between serum adenosine deaminase and total antioxidant capacity. Conclusion: Adenosine deaminase can be used as oxidative stress marker. Their increased levels indicate oxidative stress in type 2 diabetes mellitus. Therefore, estimation of serum adenosine deaminase levels help in early prediction and prevention of long term complications occurring due to oxidative stress in diabetics, thereby decreasing the mortality and morbidity in them. [Int J Res Med Sci 2015; 3(5.000: 1195-1198

  7. Adenosine concentrations in the interstitium of resting and contracting human skeletal muscle

    Hellsten, Ylva; Maclean, D.; Rådegran, G.


    BACKGROUND: Adenosine has been proposed to be a locally produced regulator of blood flow in skeletal muscle. However, the fundamental questions of to what extent adenosine is formed in skeletal muscle tissue of humans, whether it is present in the interstitium, and where it exerts its vasodilator...... and demonstrates that adenosine and its precursors increase in the exercising muscle interstitium, at a rate associated with intensity of muscle contraction and the magnitude of muscle blood flow.......BACKGROUND: Adenosine has been proposed to be a locally produced regulator of blood flow in skeletal muscle. However, the fundamental questions of to what extent adenosine is formed in skeletal muscle tissue of humans, whether it is present in the interstitium, and where it exerts its vasodilatory...... effect remain unanswered. METHODS AND RESULTS: The interstitial adenosine concentration was determined in the vastus lateralis muscle of healthy humans via dialysis probes inserted in the muscle. The probes were perfused with buffer, and the dialysate samples were collected at rest and during graded knee...

  8. Striatal adenosine signaling regulates EAAT2 and astrocytic AQP4 expression and alcohol drinking in mice.

    Lee, Moonnoh R; Ruby, Christina L; Hinton, David J; Choi, Sun; Adams, Chelsea A; Young Kang, Na; Choi, Doo-Sup


    Adenosine signaling is implicated in several neuropsychiatric disorders, including alcoholism. Among its diverse functions in the brain, adenosine regulates glutamate release and has an essential role in ethanol sensitivity and preference. However, the molecular mechanisms underlying adenosine-mediated glutamate signaling in neuroglial interaction remain elusive. We have previously shown that mice lacking the ethanol-sensitive adenosine transporter, type 1 equilibrative nucleoside transporter (ENT1), drink more ethanol compared with wild-type mice and have elevated striatal glutamate levels. In addition, ENT1 inhibition or knockdown reduces glutamate transporter expression in cultured astrocytes. Here, we examined how adenosine signaling in astrocytes contributes to ethanol drinking. Inhibition or deletion of ENT1 reduced the expression of type 2 excitatory amino-acid transporter (EAAT2) and the astrocyte-specific water channel, aquaporin 4 (AQP4). EAAT2 and AQP4 colocalization was also reduced in the striatum of ENT1 null mice. Ceftriaxone, an antibiotic compound known to increase EAAT2 expression and function, elevated not only EAAT2 but also AQP4 expression in the striatum. Furthermore, ceftriaxone reduced ethanol drinking, suggesting that ENT1-mediated downregulation of EAAT2 and AQP4 expression contributes to excessive ethanol consumption in our mouse model. Overall, our findings indicate that adenosine signaling regulates EAAT2 and astrocytic AQP4 expressions, which control ethanol drinking in mice.

  9. Striatal pre- and postsynaptic profile of adenosine A(2A receptor antagonists.

    Marco Orru

    Full Text Available Striatal adenosine A(2A receptors (A(2ARs are highly expressed in medium spiny neurons (MSNs of the indirect efferent pathway, where they heteromerize with dopamine D(2 receptors (D(2Rs. A(2ARs are also localized presynaptically in cortico-striatal glutamatergic terminals contacting MSNs of the direct efferent pathway, where they heteromerize with adenosine A(1 receptors (A(1Rs. It has been hypothesized that postsynaptic A(2AR antagonists should be useful in Parkinson's disease, while presynaptic A(2AR antagonists could be beneficial in dyskinetic disorders, such as Huntington's disease, obsessive-compulsive disorders and drug addiction. The aim or this work was to determine whether selective A(2AR antagonists may be subdivided according to a preferential pre- versus postsynaptic mechanism of action. The potency at blocking the motor output and striatal glutamate release induced by cortical electrical stimulation and the potency at inducing locomotor activation were used as in vivo measures of pre- and postsynaptic activities, respectively. SCH-442416 and KW-6002 showed a significant preferential pre- and postsynaptic profile, respectively, while the other tested compounds (MSX-2, SCH-420814, ZM-241385 and SCH-58261 showed no clear preference. Radioligand-binding experiments were performed in cells expressing A(2AR-D(2R and A(1R-A(2AR heteromers to determine possible differences in the affinity of these compounds for different A(2AR heteromers. Heteromerization played a key role in the presynaptic profile of SCH-442416, since it bound with much less affinity to A(2AR when co-expressed with D(2R than with A(1R. KW-6002 showed the best relative affinity for A(2AR co-expressed with D(2R than co-expressed with A(1R, which can at least partially explain the postsynaptic profile of this compound. Also, the in vitro pharmacological profile of MSX-2, SCH-420814, ZM-241385 and SCH-58261 was is in accordance with their mixed pre- and postsynaptic profile

  10. Adenosine A{sub 1} receptors in contrast media-induced renal dysfunction in the normal rat

    Liss, Per; Palm, Fredrik [Department of Diagnostic Radiology, University Hospital, 75185, Uppsala (Sweden); Department of Medical Cell Biology, University Hospital, 75185, Uppsala (Sweden); Carlsson, Per-Ola [Department of Medical Cell Biology, University Hospital, 75185, Uppsala (Sweden); Department of Medical Sciences, University Hospital, 75185, Uppsala (Sweden); Hansell, Peter [Department of Medical Cell Biology, University Hospital, 75185, Uppsala (Sweden)


    Renal vasoconstriction with resultant tissue hypoxia, especially in the renal medulla, has been suggested to play a role in contrast media (CM)-induced nephropathy. In this study we investigated the effects of injection of the non-ionic low-osmolar CM iopromide with and without pretreatment with the selective adenosine A{sub 1}-receptor antagonist DPCPX. The effects were evaluated on regional renal blood flow, outer medullary oxygen tension (PO{sub 2}) and urine output in normal anaesthetised rats. A laser-Doppler technique was used for recording haemodynamic changes while oxygen microelectrodes were used for oxygen measurements. The A{sub 1}-receptor antagonist per se elevated glomerular filtration rate (+44%), cortical blood flow (+15%) and urine output (threefold) while reducing outer medullary PO{sub 2} (-24%). Administration of CM reduced outer medullary blood flow (OMBF; -26%) and PO{sub 2} (-80%) but did not affect cortical blood flow. Urine output increased 28-fold by CM while arterial blood pressure was reduced. The CM-mediated effect on haemodynamics, PO{sub 2}, urine output and blood pressure was unaffected by the A{sub 1}-receptor antagonist. Adenosine A{sub 1}-receptors are not important mediators of the depression of outer medullary blood flow and PO{sub 2} caused by the CM iopromide in the normal rat; however, A{sub 1}-receptors are tonically active to regulate renal haemodynamics, PO{sub 2} and urine production during normal physiological conditions. (orig.)

  11. Diagnostic value of interferon gamma and adenosine deaminase for tuberculous pleural effusion

    Hou-rongCai; Chen-hongSun; Lin-juenDai; Zai-rongCheng


    To explore the significance of interferon gamma(IFN-γ) and adenosine deaminase (ADA)in differential diagnosis of pleural effusions. Methods: Levels of IFN-γ was measured by enzyme-linked immunosorbent assay, ADA activity was measured by colorimetric method. 37 patients with tuberculous pleural effusion and 36 patients with non-tuberculous pleurai effusions including 25 patients with malignant pleural effusions and 8 patients with pleural transudates were studied. Results: The levels of IFN-γ in patients with tuberculous pleural effusions(490.83±384.67 pg.mL-1) were higher than those with malignant pleural effusions(36.40±90.85 pg. mL-1) and pleural transudates(14.87±5.96 pg. mL-1) (P<0.01). Mean ADA activity was 52.69±17.78 U. L-1 in tuberculous pleural effusion; 19.53±13.59 in malignant pleural effusions; 9.43±4.06 inpleural transudates. The difference is significant (P<0.001). The diagnostic sensitivity of IFN-γ for tuberculous pleural effusions is 81%, specifity is 97%, the over accuracy is 90.4%. The diagnostic efficiency of ADA as following: sensitivity 89%, specifity 97%, and the over accuracy 94.5%. Conclusions: Assessments of IFN-γ and ADA in pleural effusions are of clinically diagnostic value in distinguishing tuberculous from non-tuberculous pleural effusions.

  12. Influence of hemodialysis on the plasma concentration of adenosine deaminase in patients with chronic kidney disease

    Eduardo O. Chielle


    Full Text Available ABSTRACT Introduction: Over the past years there has been a significant increase in hospitalizations and treatments due to kidney complications that eventually resulted in the increased number of patients on dialysis. The adenosine deaminase (ADA enzyme mediates the formation of some defense cells of the organism and is therefore a marker of inflammation. Objective: The objective of this study was to evaluate biomarkers of renal function and serum ADA of hemodialysis patients. Materials and methods: Blood samples were collected from 80 patients – 40 women and 40 men – between 19 and 60 years old, before and after the completion of hemodialysis. Results: There was a significant difference in levels of creatinine, urea and ADA in pre- and post-hemodialysis periods (p < 0.0001. There was a significant increase in post-dialysis ADA regardless of sex; however there was a significantly greater increase in men. Conclusion: The results showed a reduction in urea and creatinine parameters, evidencing the main purpose of hemodialysis. This study suggests that the determination of ADA activity could be used to monitor inflammation in hemodialysis patients, however wider and more specific studies are needed to show the effectiveness of serum ADA activity as an inflammatory marker in patients with chronic kidney disease.

  13. Site-directed mutagenesis and bacterial expression of human adenosine deaminase

    Danton, M.J.; Leonardo, J.; Riley, L.; Coleman, M.S.


    Adenosine deaminase (ADA) is a purine salvage pathway enzyme, the absence of which is associated with severe combined immunodeficiency disease. Time-resolved fluorescence studies, in the presence of enzyme inhibitors, indicate that at least one of the four tryptophans present in the protein molecule is close to (or in) the active site. To investigate the role of these tryptophan residues in enzyme function, they have cloned ADA cDNA into a vector in which expression is directed by the lambda P/sub R/ promoter. E. coli cells deficient in ADA were transformed with the vector construct and were shown to synthesize catalytically active human ADA. Site directed mutagenesis, coupled with a uracil selection technique for generating mutants with high efficiency, was used to construct mutant alleles of the cloned ADA. Eight mutants were obtained with base substitutions converting each of the four tryptophans to arginine or glycine. The correlation between these specific mutations and the functional expression of ADA has been examined in the ADA deficient bacterial host.

  14. Cyclic adenosine monophosphate signal pathway in targeted therapy of lymphoma

    DOU Ai-xia; WANG Xin


    Objective To review the role of cyclic adenosine monophosphate (cAMP) signal pathway in the pathogenesis oflymphoma and explore a potential lymphoma therapy targeted on this signaling pathway.Data sources The data cited in this review were mainly obtained from the articles listed in Medline and PubMed,published from January 1995 to June 2009. The search terms were "cAMP" and "lymphoma".Study selection Articles regarding the role of the cAMP pathway in apoptosis of lymphoma and associated cells and itspotential role in targeted therapy of lymphoma.Results In the transformation of lymphocytic malignancies, several signal pathways are involved. Among of them, thecAMP pathway has attracted increasing attention because of its apoptosis-inducing role in several lymphoma cells. cAMPpathway impairment is found to influence the prognosis of lymphoma. Targeted therapy to the cAMP pathway seems tobe a new direction for lymphoma treatment, aiming at restoring the cAMP function.Conclusions cAMP signal pathway has different effects on various lymphoma cells. cAMP analogues andphosphodiesterase 4B (PDE4B) inhibitors have potential clinical significance. However, many challenges remain inunderstanding the various roles of such agents.

  15. Arginine 199 and leucine 208 have key roles in the control of adenosine A2A receptor signalling function.

    Nicolas Bertheleme

    Full Text Available One successful approach to obtaining high-resolution crystal structures of G-protein coupled receptors is the introduction of thermostabilising mutations within the receptor. This technique allows the generation of receptor constructs stabilised into different conformations suitable for structural studies. Previously, we functionally characterised a number of mutants of the adenosine A2A receptor, thermostabilised either in an agonist or antagonist conformation, using a yeast cell growth assay and demonstrated that there is a correlation between thermostability and loss of constitutive activity. Here we report the functional characterisation of 30 mutants intermediate between the Rag23 (agonist conformation mutant and the wild-type receptor using the same yeast signalling assay with the aim of gaining greater insight into the role individual amino acids have in receptor function. The data showed that R199 and L208 have important roles in receptor function; substituting either of these residues for alanine abolishes constitutive activity. In addition, the R199A mutation markedly reduces receptor potency while L208A reduces receptor efficacy. A184L and L272A mutations also reduce constitutive activity and potency although to a lesser extent than the R199A and L208A. In contrast, the F79A mutation increases constitutive activity, potency and efficacy of the receptor. These findings shed new light on the role individual residues have on stability of the receptor and also provide some clues as to the regions of the protein responsible for constitutive activity. Furthermore, the available adenosine A2A receptor structures have allowed us to put our findings into a structural context.

  16. 腺苷与肺动脉高压关系研究进展%Study progress of adenosine in pulmonary arterial hypertension

    钱国清; 王良兴


    Adenosine is an endogenous nucleoside that mediates several important functions by the activation of four known cell surface (A1, A2A,A2B and A3) receptors. Adenosine therapy for the hypoxia, postoperative and other causes of pulmonary arterial hypertension has a significant effect,but its mechanisms are still controversial. This review describes the recent advances of adenosine in the study of mechanism and treatment of pulmonary arterial hypertension.%腺苷是一种内皮源性嘌呤核苷,通过与A1、A2A、A2B及A3四种腺苷受体亚型相结合而发挥不同的生物学效应.腺苷对于缺氧、术后和特发性等多种原因引起的肺动脉高压都具有明显的治疗效果,但其作用机制尚存在争议.本文综述了腺苷对肺动脉高压的作用机制及治疗研究进展.

  17. A genetic variation in the adenosine A2A receptor gene (ADORA2A) contributes to individual sensitivity to caffeine effects on sleep.

    Rétey, J V; Adam, M; Khatami, R; Luhmann, U F O; Jung, H H; Berger, W; Landolt, H-P


    Caffeine is the most widely used stimulant in Western countries. Some people voluntarily reduce caffeine consumption because it impairs the quality of their sleep. Studies in mice revealed that the disruption of sleep after caffeine is mediated by blockade of adenosine A2A receptors. Here we show in humans that (1) habitual caffeine consumption is associated with reduced sleep quality in self-rated caffeine-sensitive individuals, but not in caffeine-insensitive individuals; (2) the distribution of distinct c.1083T>C genotypes of the adenosine A2A receptor gene (ADORA2A) differs between caffeine-sensitive and -insensitive adults; and (3) the ADORA2A c.1083T>C genotype determines how closely the caffeine-induced changes in brain electrical activity during sleep resemble the alterations observed in patients with insomnia. These data demonstrate a role of adenosine A2A receptors for sleep in humans, and suggest that a common variation in ADORA2A contributes to subjective and objective responses to caffeine on sleep.

  18. Nucleus accumbens neurotransmission and effort-related choice behavior in food motivation: effects of drugs acting on dopamine, adenosine, and muscarinic acetylcholine receptors.

    Nunes, Eric J; Randall, Patrick A; Podurgiel, Samantha; Correa, Mercè; Salamone, John D


    Mesolimbic dopamine (DA) is a critical component of the brain circuitry regulating behavioral activation and effort-related processes. Although nucleus accumbens (NAc) DA depletions or antagonism leave aspects of appetite and primary food motivation intact, rats with impaired DA transmission reallocate their instrumental behavior away from food-reinforced tasks with high response requirements, and instead select less effortful food-seeking behaviors. Previous work showed that adenosine A2A antagonists can reverse the effects of DA D2 antagonists on effort-related choice, and that stimulation of adenosine A2A receptors produces behavioral effects that are similar to those induced by DA antagonism. The present review summarizes the literature on the role of NAc DA and adenosine in effort-related processes, and also presents original data on the effects of local stimulation of muscarinic acetylcholine receptors in NAc core. Local injections of the muscarinic agonist pilocarpine directly into NAc core produces shifts in effort-related choice behavior similar to those induced by DA antagonism or A2A receptor stimulation, decreasing lever pressing but increasing chow intake in rats responding on a concurrent fixed ratio/chow feeding choice task. In contrast, injections into a neostriatal control site dorsal to the NAc were ineffective. The actions of pilocarpine on this task were attenuated by co-administration of the muscarinic antagonist scopolamine. Thus, drugs that act on DA, adenosine A2A, and muscarinic receptors regulate effort-related choice behavior, which may have implications for the treatment of psychiatric symptoms such as psychomotor slowing, fatigue or anergia that can be observed in depression and other disorders.

  19. Adenosine A2A receptor and ecto-5'-nucleotidase/CD73 are upregulated in hippocampal astrocytes of human patients with mesial temporal lobe epilepsy (MTLE).

    Barros-Barbosa, Aurora R; Ferreirinha, Fátima; Oliveira, Ângela; Mendes, Marina; Lobo, M Graça; Santos, Agostinho; Rangel, Rui; Pelletier, Julie; Sévigny, Jean; Cordeiro, J Miguel; Correia-de-Sá, Paulo


    Refractoriness to existing medications of up to 80 % of the patients with mesial temporal lobe epilepsy (MTLE) prompts for finding new antiepileptic drug targets. The adenosine A2A receptor emerges as an interesting pharmacological target since its excitatory nature partially counteracts the dominant antiepileptic role of endogenous adenosine acting via inhibitory A1 receptors. Gain of function of the excitatory A2A receptor has been implicated in a significant number of brain pathologies commonly characterized by neuronal excitotoxicity. Here, we investigated changes in the expression and cellular localization of the A2A receptor and of the adenosine-generating enzyme, ecto-5'-nucleotidase/CD73, in the hippocampus of control individuals and MTLE human patients. Western blot analysis indicates that the A2A receptor is more abundant in the hippocampus of MTLE patients compared to control individuals. Immunoreactivity against the A2A receptor predominates in astrocytes staining positively for the glial fibrillary acidic protein (GFAP). No co-localization was observed between the A2A receptor and neuronal cell markers, like synaptotagmin 1/2 (nerve terminals) and neurofilament 200 (axon fibers). Hippocampal astrogliosis observed in MTLE patients was accompanied by a proportionate increase in A2A receptor and ecto-5'-nucleotidase/CD73 immunoreactivities. Given our data, we hypothesize that selective blockade of excessive activation of astrocytic A2A receptors and/or inhibition of surplus adenosine formation by membrane-bound ecto-5'-nucleotidase/CD73 may reduce neuronal excitability, thus providing a novel therapeutic target for drug-refractory seizures in MTLE patients.

  20. Feasibility and safety of high-dose adenosine perfusion cardiovascular magnetic resonance

    Holloway Cameron J


    Full Text Available Abstract Introduction Adenosine is the most widely used vasodilator stress agent for Cardiovascular Magnetic Resonance (CMR perfusion studies. With the standard dose of 140 mcg/kg/min some patients fail to demonstrate characteristic haemodynamic changes: a significant increase in heart rate (HR and mild decrease in systolic blood pressure (SBP. Whether an increase in the rate of adenosine infusion would improve peripheral and, likely, coronary vasodilatation in those patients is unknown. The aim of the present study was to assess the tolerance and safety of a high-dose adenosine protocol in patients with inadequate haemodynamic response to the standard adenosine protocol when undergoing CMR perfusion imaging. Methods 98 consecutive patients with known or suspected coronary artery disease (CAD underwent CMR perfusion imaging at 1.5 Tesla. Subjects were screened for contraindications to adenosine, and an electrocardiogram was performed prior to the scan. All patients initially received the standard adenosine protocol (140 mcg/kg/min for at least 3 minutes. If the haemodynamic response was inadequate (HR increase Results All patients successfully completed the CMR scan. Of a total of 98 patients, 18 (18% did not demonstrate evidence of a significant increase in HR or decrease in SBP under the standard adenosine infusion rate. Following the increase in the rate of infusion, 16 out of those 18 patients showed an adequate haemodynamic response. One patient of the standard infusion group and two patients of the high-dose group developed transient advanced AV block. Significantly more patients complained of chest pain in the high-dose group (61% vs. 29%, p = 0.009. On multivariate analysis, age > 65 years and ejection fraction Conclusions A substantial number of patients do not show adequate peripheral haemodynamic response to standard-dose adenosine stress during perfusion CMR imaging. Age and reduced ejection fraction are predictors of inadequate